Agrobacterium-mediated Genetic Transformation Research Articles

Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower

Sunflower is one of the four major oil crops in the world. ‘Zaoaidatou’ (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.

Visualization of mitochondrial dynamics in tomato based on green fluorescent protein

This study aimed to visualize the morphological features and dynamic changes of tomato mitochondria to provide a basis for the study of its mitochondrial functions. In this study, transgenic tomatoes expressing mitochondria-localized green fluorescent protein (mitochondria-GFP, Mt-GFP) were obtained by Agrobacterium-mediated genetic transformation. The color, hardness, soluble solids, acidity content, respiration rate, and ethylene production of the transgenic Mt-GFP tomato fruits were determined at the stage of mature green, breaker, and 3, 6, 9 days after breaker, while the wild-type tomato fruits were used as a control. As expected, Mt-GFP recombinant protein did not affect the ripening process, but induced the increased acidity of tomato fruits. The accumulations of Mt-GFP protein in tomato leaves and fruits were successfully verified by Western blotting. The morphological characteristics of mitochondria in flower, leaf and fruit cells as well as the dynamic changes of mitochondria in flower cells were clearly observed and studied under confocal laser microscope. The development of transgenic Mt-GFP tomato plants helps the visualization of tomato mitochondria and provides good research materials for the study of mitochondrial function during tomato development and fruit ripening.

Overexpression of SQUAMOSA promoter binding protein-like 4a (NtSPL4a) alleviates Cd toxicity in Nicotiana tabacum

Squamosa Promoter Binding Protein-Like (SPL) plays a crucial role in regulating plant development and combating stress, yet its mechanism in regulating resistance to Cd toxicity remains unclear. In this study, we cloned a nuclear-localized transcription factor, NtSPL4a, from the tobacco cultivar TN90. Transient co-expression results showed that miR156 significantly reduced the expression of NtSPL4a by binding to the 3′-UTR of its transcript. We obtained transgenic tobacco overexpressing NtSPL4a (including the 3′-UTR) and NtSPL4aΔ (lacking the 3′-UTR) through Agrobacterium-mediated genetic transformation. Compared to the wild type (WT), overexpression of NtSPL4a/NtSPL4aΔ shortened the flowering time and exhibited a more developed root system. The transgenic tobacco showed significantly reduced Cd content, being 85.1% (OE-NtSPL4a) and 46.7% (OE-NtSPL4aΔ) of WT, respectively. Moreover, the upregulation of NtSPL4a affected the mineral nutrient homeostasis in transgenic tobacco. Additionally, overexpression of NtSPL4a/NtSPL4aΔ effectively alleviated leaf chlorosis and oxidative stress induced by Cd toxicity. One possible reason is that the overexpression of NtSPL4a/NtSPL4aΔ can effectively promote the accumulation of non-enzymatic antioxidants. A comparative transcriptomic analysis was performed between transgenic tobacco and WT to further unravel the global impacts brought by NtSPL4a. The tobacco overexpressing NtSPL4a had 183 differentially expressed genes (77 upregulated, 106 downregulated), while the tobacco overexpressing NtSPL4aΔ had 594 differentially expressed genes (244 upregulated, 350 downregulated) compared to WT. These differentially expressed genes mainly included transcription factors, metal transport proteins, flavonoid biosynthesis pathway genes, and plant stress-related genes. Our study provides new insights into the role of the transcript factor SPL in regulating Cd tolerance.

Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis)

Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis)

Agrobacterium-mediated Genetic Transformation and Plant Regeneration from Cotyledons in Philippine Eggplant (Solanum melongena L.) Acc. ‘PH 11424’

Agrobacterium-mediated Genetic Transformation and Plant Regeneration from Cotyledons in Philippine Eggplant (Solanum melongena L.) Acc. ‘PH 11424’

Manifestation of agronomically valuable traits in the progeny of a sorghum mutant carrying the genetic construct for RNA silencing of the γ-kafirin gene.

Improving the nutritional value of grain sorghum, a drought- and heat-tolerant grain crop, is an important task in the context of global warming. One of the reasons for the low nutritional value of sorghum grain is the resistance of its storage proteins (kafirins) to proteolytic digestion, which is due, among other things, to the structural organization of protein bodies, in which γ-kafirin, the most resistant to proteases, is located on the periphery, encapsulating more easily digested α-kafirins. The introduction of genetic constructs capable of inducing RNA silencing of the γ-kafirin (gKAF1) gene opens up prospects for solving this problem. Using Agrobacterium-mediated genetic transformation of immature embryos of the grain sorghum cv. Avans we have obtained a mutant with improved digestibility of endosperm proteins (up to 92 %) carrying a genetic construct for RNA silencing of the gKAF1 gene. The goal of this work was to study the stability of inheritance of the introduced genetic construct in T2-T4 generations, to identify the number of its copies, as well as to trace the manifestation of agronomically valuable traits in the offspring of the mutant. The mutant lines were grown in experimental plots in three randomized blocks. The studied lines were characterized by improved digestibility of kafirins, a modified type of endosperm, completely or partially devoid of the vitreous layer, an increased percentage of lysine (by 75 %), reduced plant height, peduncle length, 1000-grains weight, and grain yield from the panicle. In T2, a line with monogenic control of GA resistance was selected. qPCR analysis showed that in different T3 and T4 plants, the genetic construct was present in 2-4 copies. In T3, a line with a high digestibility of endosperm proteins (81 %) and a minimal decrease in agronomically valuable traits (by 5-7 %) was selected.

Establishment of a Rapid and Effective Agrobacterium-Mediated Genetic Transformation System of Oxalis triangularis 'Purpurea'.

Oxalis triangularis 'Purpurea' has significant ornamental value in landscaping. There is a critical necessity to elucidate the gene functions of O. triangularis 'Purpurea' and dissect the molecular mechanisms governing key ornamental traits. However, a reliable genetic transformation method remains elusive. In this study, our investigation revealed that various transformation parameters, including recipient material (petioles), pre-culture time (2-5 days), acetosyringone (AS) concentration (100-400 μM), Agrobacterium concentrations (OD600 = 0.4-1.0), infection time (5-20 min), and co-culture time (2-5 days), significantly impacted the stable genetic transformation in O. triangular 'Purpurea'. Notably, the highest genetic transformation rate was achieved from the leaf discs pre-cultured for 3 days, treated with 200 μM AS infected with Agrobacterium for 11 min at OD600 of 0.6, and subsequently co-cultured for 3 days. This treatment resulted in a genetic transformation efficiency of 9.88%, and it only took 79 days to produce transgenic plants. Our transformation protocol offers advantages of speed, efficiency, and simplicity, which will greatly facilitate genetic transformation for O. triangular 'Purpurea' and gene function studies.

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.

Agrobacterium-Mediated Genetic Transformation of Withania coagulans (Dunal) with rol A Genes and Its Antioxidant Potential.

In ancient times, Withania coagulans Dunal was used as a therapeutic plant for the treatment of several diseases. This report aims to examine the effect of Agrobacterium tumefactions-mediated transformation of W. coagulans with the rolA gene to enhance secondary metabolite production, antioxidant activity, and anticancer activity of transformed tissues. Before transgenic plant production, the authors designed an efficient methodology for in vitro transformation. In this study, leaf explants were cultured on Murashage and Skoog (MS) media containing different amounts of naphthalene acetic acid (NAA) and benzyl adenine (BA). The best performance for inducing embryogenic callus was in MS medium containing 4 μM NAA and 6.0 μM BA, while the best results for shooting (100%) were obtained at 8 μM benzyl adenine. On the other hand, direct shooting was attained by subculturing leaves on MS medium supplemented with 8 μM benzyl adenine. Prolonged shoots showed excellent in vitro rooting results (80%) with 12 μM indole-3-butyric acid (IBA). The samples were precultivated for 3 days and were followed by 48 h infection with A. tumefaciens strain GV3101 having pCV002. Then, a vector expressed the rol A gene of strain Agrobacterium rhizogenes. Furthermore, three independent transgenic shoot lines and one callus line (T2) were produced and exhibited stable integration of transgene rol A genes, as revealed by PCR analysis. Transgenic strains showed a significant increase in antioxidant potential as compared to untransformed plants. Additionally, LC-MS analysis showed that the transformed strains have a higher withanolide content as compared to untransformed ones. Moreover, the reduced proliferation of prostate cancer cells was observed after treatment with extracts of transgenic plants. Furthermore, these transformed plants exhibited superior antioxidant capability and higher withanolide content than untransformed ones. In conclusion, the reported data can be used to select withanolide-rich germplasm from transformed cell cultures.

Enhancing drought resistance in Pogostemon cablin (Blanco) Benth. through overexpression of ACC deaminase gene using thin cell layer regeneration system.

Pogostemon cablin cultivation faces massive constraints because of its susceptability to drought stress that reduces patchouli propagation and oil yield. The present study has achieved an efficient and rapid direct regeneration system for the transgenic production of P. cablin using Agrobacterium-mediated genetic transformation. To establish an efficient regeneration protocol for fast in-vitro multiplication of patchouli plants, leaf, petiole, and transverse thin cell layer (tTCL) explants were used and inoculated on an MS medium supplemented with different combinations of phytohormones. A comparative study showed a maximum regeneration frequency of 93.30 ± 0.56% per explant was obtained from leaf segments on optimal MS medium fortified with 0.2mg/L BAP and 0.1mg/L NAA. Leaf and petiole explants took 25-35 days to regenerate while tTCL section showed regeneration in just 15-20 days on the same medium. Subsequently, productive genetic transformation protocol OD600 0.6, AS 200µM, 30mg/L kanamycin, and infection time 5 min. was standardized and best-suited explants were infected at optimum conditions from the Agrobacterium tumefaciens (LBA 4404) strain harboring ACC deaminase to generate transgenic P. cablin Benth. (CIM-Samarth) plants. The investigation suggested that the optimized protocol provides a maximum transformation frequency of 42 ± 1.9% in 15-20 days from tTCL. The transgenic plants were shifted to the greenhouse with a 52.0 ± 0.8% survival frequency. A molecular docking study confirmed significant binding affinity of ligand ACC with ACC deaminase at the catalytic site, and ligand interactions showed four H-bonds at the binding pocket with amino acids Cys-196, Val-198, Thr-199, and Gly-200 that validate gene relative expression in transgenic plants. Among all transgenic acclimatized greenhouse-grown patchouli plants, line PT4 showed improved drought resistance under severe water stress as its RWC was 71.7 ± 2.3% to 75.7 ± 2.1% which is greater than the RWC of the control plant, 58.30 ± 0.21%. Analysis of the other physiological indicators, H2O2, chlorophyll content, and ROS result support drought resistance ability. Our study concluded that the first report on P. cablin, tTCL direct regeneration, and standardized transformation protocol created a new opportunity for genetic manipulation to achieve drought-resistant patchouli plants for cultivation in all seasons at the commercial level.

Prediction and optimization of indirect shoot regeneration of Passiflora caerulea using machine learning and optimization algorithms

BackgroundOptimization of indirect shoot regeneration protocols is one of the key prerequisites for the development of Agrobacterium-mediated genetic transformation and/or genome editing in Passiflora caerulea. Comprehensive knowledge of indirect shoot regeneration and optimized protocol can be obtained by the application of a combination of machine learning (ML) and optimization algorithms.Materials and methodsIn the present investigation, the indirect shoot regeneration responses (i.e., de novo shoot regeneration rate, the number of de novo shoots, and length of de novo shoots) of P. caerulea were predicted based on different types and concentrations of PGRs (i.e., TDZ, BAP, PUT, KIN, and IBA) as well as callus types (i.e., callus derived from different explants including leaf, node, and internode) using generalized regression neural network (GRNN) and random forest (RF). Moreover, the developed models were integrated into the genetic algorithm (GA) to optimize the concentration of PGRs and callus types for maximizing indirect shoot regeneration responses. Moreover, sensitivity analysis was conducted to assess the importance of each input variable on the studied parameters.ResultsThe results showed that both algorithms (RF and GRNN) had high predictive accuracy (R2 > 0.86) in both training and testing sets for modeling all studied parameters. Based on the results of optimization process, the highest de novo shoot regeneration rate (100%) would be obtained from callus derived from nodal segments cultured in the medium supplemented with 0.77 mg/L BAP plus 2.41 mg/L PUT plus 0.06 mg/L IBA. The results of the sensitivity analysis showed the explant-dependent impact of exogenous application of PGRs on indirect de novo shoot regeneration.ConclusionsA combination of ML (GRNN and RF) and GA can display a forward-thinking aid to optimize and predict in vitro culture systems and consequentially cope with several challenges faced currently in Passiflora tissue culture.

Cloning and characterization of the promoters of the key genes CPT, SRPP and REF involved in Periploca sepium rubber biosynthesis

Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1, 4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (β-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.

Optimised Agrobacterium-Mediated Transformation and Application of Developmental Regulators Improve Regeneration Efficiency in Melons.

Melon (Cucumis melo L.) is a protected crop in China with high economic value. Agrobacterium-mediated genetic transformation is a powerful tool to improve agronomic traits and obtain elite germplasm. However, current transformation protocols in melons are inefficient and highly genotype-dependent. To improve transformation in melon, we tested different infiltration methods for Agrobacterium-mediated transformation. Among these methods, micro-brushing and sonication for 20 s, followed by vacuum infiltration at -1.0 kPa for 90 s, resulted in the strongest green fluorescent protein signal and increased the proportion of infected explants. We transformed melon with developmental regulatory genes AtGRF5, AtPLT5, AtBBM, AtWUS, AtWOX5, and AtWIND1 from Arabidopsis and estimated regeneration frequencies as the number of regenerating shoots/total number of inoculated explants in the selection medium. The overexpression of AtGRF5 and AtPLT5 in melon resulted in transformation efficiencies of 42.3% and 33% in ZHF and 45.6% and 32.9% in Z12, respectively, which were significantly higher than those of the control. AtGRF5 and AtPLT5 expression cassettes were added to CRISPR/Cas9 genome-editing vectors to obtain transgenic phytoene desaturase CmPDS knockout mutants. Using AtGRF5 or AtPLT5, multi-allelic mutations were observed at CmPDS target sites in recalcitrant melon genotypes. This strategy enables genotype-flexible transformation and promotes precise genome modification technologies in melons.

Agrobacterium-mediated Genetic Transformation of two Peanut (Arachis hypogaea L.) Varieties using Salinity Tolerant PDH45 Gene

An efficient Agrobacterium-mediated genetic transformation protocol has been developed in the present investigation to integrate salinity tolerant gene pea DNA helicase (PDH45) in two peanut varieties, Dhaka-1 and BARI Chinabadam-8. Decapitated half-embryo explants of the above-mentioned varieties were also used to develop a transformation compatible in vitro regeneration system. MS medium supplemented with 5.0 mg/l BAP and 0.5 mg/l Kn showed maximum response for shoot induction in both varieties. Half strength of MS medium supplemented with 0.2 mg/l IBA showed better response towards root induction for both varieties. Genetic transformation experiments were performed using Agrobacterium strain LBA4404 containing pCAMBIA1301-PDH45 conferring reporter gus gene, hygromycin resistance hpt gene, and salinity tolerant gene pdh45. The transformation variables were optimized, OD600 1.4 with 45 minutes of incubation period, and three days of co-culture showed the best performance. The transformed shoots were selected using 30 mg/l hygromycin. The overall transformation frequency was very low, where 0.10% shoots of Dhaka-1 and 0.07% shoots of BARI Chinabadam-8 survived the selection pressure. The stable integration of the transgene in the transformed shoots was confirmed through PCR analysis. Plant Tissue Cult. & Biotech. 33(1): 37-46, 2023 (June)

Barley stripe mosaic virus-mediated somatic and heritable gene editing in barley (Hordeum vulgare L.).

Genome editing strategies in barley (Hordeum vulgare L.) typically rely on Agrobacterium-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants that constitutively express Cas9. Here, we explored virus-induced genome editing (VIGE) based on barley stripe mosaic virus (BSMV) in Cas9-transgenic barley. Somatic and heritable editing in the ALBOSTRIANS gene (CMF7) resulting in albino/variegated chloroplast-defective barley mutants is shown. In addition, somatic editing in meiosis-related candidate genes in barley encoding ASY1 (an axis-localized HORMA domain protein), MUS81 (a DNA structure-selective endonuclease), and ZYP1 (a transverse filament protein of the synaptonemal complex) was achieved. Hence, the presented VIGE approach using BSMV enables rapid somatic and also heritable targeted gene editing in barley.

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.

An efficient and optimized protocol for tall fescue tissue culture and Agrobacterium-mediated genetic transformation

An efficient and optimized protocol for tall fescue tissue culture and Agrobacterium-mediated genetic transformation

MDSi: Multi-omics Database for Setaria italica

BackgroundFoxtail millet (Setaria italica) harbors the small diploid genome (~ 450 Mb) and shows the high inbreeding rate and close relationship to several major foods, feed, fuel and bioenergy grasses. Previously, we created a mini foxtail millet, xiaomi, with an Arabidopsis-like life cycle. The de novo assembled genome data with high-quality and an efficient Agrobacterium-mediated genetic transformation system made xiaomi an ideal C4 model system. The mini foxtail millet has been widely shared in the research community and as a result there is a growing need for a user-friendly portal and intuitive interface to perform exploratory analysis of the data.ResultsHere, we built a Multi-omics Database for Setaria italica (MDSi, http://sky.sxau.edu.cn/MDSi.htm), that contains xiaomi genome of 161,844 annotations, 34,436 protein-coding genes and their expression information in 29 different tissues of xiaomi (6) and JG21 (23) samples that can be showed as an Electronic Fluorescent Pictograph (xEFP) in-situ. Moreover, the whole-genome resequencing (WGS) data of 398 germplasms, including 360 foxtail millets and 38 green foxtails and the corresponding metabolic data were available in MDSi. The SNPs and Indels of these germplasms were called in advance and can be searched and compared in an interactive manner. Common tools including BLAST, GBrowse, JBrowse, map viewer, and data downloads were implemented in MDSi.ConclusionThe MDSi constructed in this study integrated and visualized data from three levels of genomics, transcriptomics and metabolomics, and also provides information on the variation of hundreds of germplasm resources that can satisfies the mainstream requirements and supports the corresponding research community.

Novel Camellia sinensis O-Methyltransferase Regulated by CsMADSL1 Specifically Methylates EGCG in Cultivar “GZMe4”

Epigallocatechin-3-O-(4-O-methyl)gallate (EGCG4″Me) in Camellia sinensis possesses numerous beneficial biological activities. However, the germplasm rich in EGCG4″Me and the O-methyltransferase responsible for EGCG4″Me biosynthesis are poorly understood. Herein, the content of EGCG3″Me and EGCG4″Me in the shoots of 13 cultivars was analyzed to demonstrate that EGCG4″Me is characteristically accumulated in the "GZMe4" cultivar but not in the other 12 cultivars. A novel O-methyltransferase (CsOMTL1) was identified from "GZMe4" using RNA-Seq and correlation analysis. Using the recombinant enzyme, EGCG4″Me was synthesized in vitro. Overexpression of CsOMTL1 via Agrobacterium-mediated genetic transformation caused constitutive accumulation of EGCG4″Me in C. sinensis callus. Moreover, the transcription factor CsMADSL1 localized in the nucleus activated the transcription of CsOMTL1 and specifically interacted with its promoter. Hence, our study identified a novel O-methyltransferase that characteristically catalyzes the synthesis of EGCG4″Me and a positive regulator of EGCG4″Me synthesis in "GZMe4", which might provide a strategy for the breeding of a tea cultivar rich in EGCG4″Me.

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.