Agrobacterium-mediated Transformation System Research Articles

Establishment of an efficient regeneration and genetic transformation system for Malus prunifolia Borkh. ‘Fupingqiuzi’

Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function. However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed. Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium (MS basal salts, 30 g L−1 sucrose, 8 g L−1 agar, 5 mg L−1 6-benzylaminopurine (6-BA), 2 mg L−1 thidiazuron (TDZ), and 1 mg L−1 1-naphthlcetic acid (NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime (150 mg L−1) and kanamycin (15 mg L−1). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research.

Expression of OsDREB2A in Transgenic Tomato Improves Drought Tolerance

Dehydration responsive element binding (DREB) are important regulatory molecules which have a crucial role in abiotic stress tolerance. The productivity of tomato, as a drought-sensitive crop, is highly restricted by drought stress. The current study aimed at introducing the OsDERB2A gene into two tomato genotypes via Agrobacterium-mediated transformation system. Cotyledonary explants were pre-cultured for two days with Agrobacterium strain LBA4404 harboring pCAMBIA1301 with OsDREB2A driven by the constitutive promoter CaMV35S for transformation. Shoots were directly regenerated on MS medium containing 1 mg l-1 zeatin and 1 mg l-1 BAP, and in presence of 30 mg l-1 hygromycin as selective agent. Only eight weeks were needed to regenerate transgenic tomato using this protocol. An OD600 of 0.4 resulted in 64.3-76.9% transformation efficiency. Stable integration and expression of the OsDREB2A gene were confirmed in transgenic tomato using PCR and RT-PCR analyses, and drought tolerance of T0 transgenic lines was confirmed by leaf disc assay in 300 mM mannitol. The superior biomass, photosynthetic pigments, free soluble sugars and proline accumulation of OsDREB2A transgenic lines over wild type in response to mannitol-stress revealed their enhanced drought tolerance and indicated that the constitutive expression of OsDREB2A might modulate the expression of other drought responsive genes.

Efficient Transformation of Catalpa bungei Shows Crystal Genes Conferring Resistance to the Shoot Borer Omphisa plagialis.

Although Catalpa bungei is a forest plant with considerable economic and ornamental value in China, its wood and decorative qualities are constrained by insect pests such as the shoot borer Omphisa plagialis (Lepidoptera). Overexpressing insect resistance genes such as crystal genes to develop an insect-resistant variety of C. bungei is an environmental and ecological approach. However, genotype limitations and low regeneration rates of embryogenic calli (EC) inhibit the development of transformation and the insect-resistant gene expression system in C. bungei. Here, we first established embryogenic callus induction and regeneration systems of five genotypes using mature seed and stem segment explants; the highest induction and regeneration rates of EC were 39.89 and 100%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was developed from EC and its positive frequency was up to 92.31%. Finally, using the transformation system, 15 and 22 transgenic C. bungei lines that expressed Cry2A and Cry9Aa-like were generated, respectively. These transgenic lines that exhibited significantly higher resistance to O. plagialis in the laboratory and field have great promise for meeting the challenge of future pest management under changing climatic conditions. Additionally, this efficient, fast, and stable transformation system could be a potential tool for gene function analysis and forest tree genetic improvement.

Forward and reverse genetic dissection of morphogenesis identifies filament-competent Candida auris strains

Candida auris is an emerging healthcare-associated pathogen of global concern. Recent reports have identified C. auris isolates that grow in cellular aggregates or filaments, often without a clear genetic explanation. To investigate the regulation of C. auris morphogenesis, we applied an Agrobacterium-mediated transformation system to all four C. auris clades. We identified aggregating mutants associated with disruption of chitin regulation, while disruption of ELM1 produced a polarized, filamentous growth morphology. We developed a transiently expressed Cas9 and sgRNA system for C. auris that significantly increased targeted transformation efficiency across the four C. auris clades. Using this system, we confirmed the roles of C. auris morphogenesis regulators. Morphogenic mutants showed dysregulated chitinase expression, attenuated virulence, and altered antifungal susceptibility. Our findings provide insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic tools described here will allow for efficient manipulation of the C. auris genome.

Genetic evidence for the requirements of antroquinonol biosynthesis by Antrodia camphorata during liquid-state fermentation.

The solid-state fermentation of Antrodia camphorata could produce a variety of ubiquinone compounds, such as antroquinonol (AQ). However, AQ is hardly synthesized during liquid-state fermentation (LSF). To investigates the mechanism of AQ synthesis, three precursors (ubiquinone 0 UQ0, farnesol and farnesyl diphosphate FPP) were added in LSF. The results showed that UQ0 successfully induced AQ production; however, farnesol and FPP could not induce AQ synthesis. The precursor that restricts the synthesis of AQ is the quinone ring, not the isoprene side chain. Then, the Agrobacterium-mediated transformation system of A. camphorata was established and the genes for quinone ring modification (coq2-6) and isoprene synthesis (HMGR, fps) were overexpressed. The results showed that overexpression of genes for isoprene side chain synthesis could not increase the yield of AQ, but overexpression of coq2 and coq5 could significantly increase AQ production. This is consistent with the results of the experiment of precursors. It indicated that the A. camphorata lack the ability to modify the quinone ring of AQ during LSF. Of the modification steps, prenylation of UQ0 is the key step of AQ biosynthesis. The result will help us to understand the genetic evidence for the requirements of AQ biosynthesis in A. camphorata.

In Vivo Functional Verification of Four Related Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves.

The alkaloid 1-deoxynojirimycin (DNJ) is one of the major bioactive compounds in mulberry leaves (Morus alba L.). Previously, we discovered four key genes involved in the pathway from lysine to piperidine in the biosynthesis of DNJ in mulberry leaves, MaLDC (MG727866), MaCAO (MH205733), MaSDR1 (MT989445), and MaSDR2 (MT989446), which encoded lysine decarboxylase, copper amine oxidase, and short-chain dehydrogenase/reductase 1 and 2, respectively. However, the in vivo functions of these four genes have not been verified yet. Here, these four genes were successfully cloned and used for the establishment of C58C1 Agrobacterium rhizogenes mediated overexpression genetic transformation systems and GV3101 Agrobacterium-mediated virus-induced gene silencing transformation systems in order to verify the influence of these four genes on the biosynthetic content of DNJ in mulberry leaves. The results showed that the content of DNJ increased after the four genes were overexpressed. When these four genes were silenced, the gene expression was blocked, which affected the biosynthesis of DNJ, and the DNJ content decreased. The above results indicated that these four genes participated in DNJ biosynthesis. This study provided a foundation for further elucidating the regulatory mechanisms of DNJ biosynthesis in mulberry leaves.

Establishment of an efficient Agrobacterium-mediated genetic transformation system in halophyte Puccinellia tenuiflora.

The online version contains supplementary material available at 10.1007/s11032-021-01247-8.

An Agrobacterium-mediated non-antibiotic selection-based transformation system for rice (Oryza sativa ssp. indica) cultivar “93-11” successfully produces TAC1-silenced transgenic plants

An Agrobacterium-mediated non-antibiotic selection-based transformation system for rice (Oryza sativa ssp. indica) cultivar “93-11” successfully produces TAC1-silenced transgenic plants

Tissue Culture- and Selection-Independent Agrobacterium tumefaciens-Mediated Transformation of a Recalcitrant Grain Legume, Cowpea (Vigna unguiculata L. Walp).

A simple and generally fast Agrobacterium-mediated transformation system with no tissue culture and selection steps has been developed for the first time in a recalcitrant food legume, cowpea. The approach involves wounding of 1-day-old germinated seeds with a needle or sonication either alone or in combination of vacuum infiltration with A. tumefaciens EH105 (pCAMBIA2301) carrying a β-glucuronidase (GUS) gene (uidA) and a neomycin phosphotransferase (nptII) gene for stable transformation. Sonicated and vacuum infiltrated seedlings showed the highest transient GUS activity in 90% of the explants. The sprouted co-cultured seeds directly established in soil and without selection were allowed to develop into plants which on maturity produced T0 seeds. The presence of the alien genes, nptII and uidA in T0 plants and their integration into the genome of T1 plants were confirmed by polymerase chain reaction (PCR) and Southern blot analyses, respectively. The transgenes were inherited in the subsequent T2 generation in a Mendelian fashion and their expression was confirmed by semi-quantitative PCR. The transformation frequency of 1.90% was obtained with sonication followed by vacuum infiltration with Agrobacterium. This approach provides favorable circumstances for the rapid meristem transformation and likely makes translational research ease in an important recalcitrant food legume, cowpea.

Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos

BackgroundTrachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg’s B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. ResultsAccording to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. ConclusionsIn this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.

Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae.

Genetic engineering of the filamentous fungus Aspergillus oryzae still requires more suitable selection markers for fungal transformation. Our previous work has shown that Agrobacterium tumefaciens-mediated transformation (ATMT) based on the uridine/uracil auxotrophic mechanism with pyrG as the selection marker is very efficient for gene transfer in A. oryzae. In the present study, we delete the hisB gene, which is essential for histidine biosynthesis, in A. oryzae via homologous recombination and demonstrate that hisB is a reliable selection marker for genetic transformation of this fungus. Under optimal conditions, the ATMT efficiency of the histidine auxotrophic A. oryzae reached 515 transformants per 106 spores. Especially, we have succeeded in constructing a new ATMT system based on dual auxotrophic A. oryzae mutants with two different selection markers including hisB and pyrG. This dual auxotrophic ATMT system displayed a transformation efficiency of 232 transformants per 106 spores for the hisB marker and 318 transformants per 106 spores for the pyrG marker. By using these selectable markers, the co-expression of the DsRed and GFP fluorescent reporter genes was implemented in a single fungal strain. Furthermore, we could perform both the deletion and complementation of the laeA regulatory gene in the same strain of A. oryzae to examine its function. Conclusively, the ATMT system constructed in our work represents a promising genetic tool for studies on recombinant expression and gene function in the industrially important fungus A. oryzae.

Establishment of a new and efficient Agrobacterium-mediated transformation system in the nematicidal fungus Purpureocillium lilacinum

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is widely commercialized for controlling plant-parasitic nematodes and represents a potential cell factory for enzyme production. This nematicidal fungus is intrinsically resistant to common antifungal agents used for genetic transformation. Therefore, molecular investigations in P. lilacinum are still limited so far. In the present study, we have established a new Agrobacterium tumefaciens-mediated transformation (ATMT) system in P. lilacinum based on the uridine/uracil auxotrophic mechanism. Here, uridine/uracil auxotrophic mutants were simply generated via UV irradiation instead of a complicated genetic approach for the pyrG gene deletion. A stable uridine/uracil auxotrophic mutant was then selected as a recipient for fungal transformation. We further indicated that the pyrG gene from Aspergillus niger can be used as a selectable marker for genetic transformation of P. lilacinum. Under optimized conditions for ATMT, the transformation efficiency reached 2873 ± 224 transformants per 106 spores. Using the constructed ATMT system, we succeeded in expressing the DsRed reporter gene in P. lilacinum. Additionally, we have identified a very promising mutant for chitinase production from a collection of T-DNA insertion transformants. This mutant possesses a special phenotype of hyper-branching mycelium and produces more conidia in comparison to the wild strain. Conclusively, our ATMT system can be exploited for overexpression of target genes or for T-DNA insertion mutagenesis in the agriculturally important fungus P. lilacinum. The genetic approach in the present work may also be applied for developing similar ATMT systems in other fungi, especially for fungi that their genome databases are currently not available.

Overexpression of a Rosa rugosa Thunb. NUDX gene enhances biosynthesis of scent volatiles in petunia.

Rosa rugosa is an important natural perfume plant in China. Rose essential oil is known as ‘liquid gold’ and has high economic and health values. Monoterpenes are the main fragrant components of R. rugosa flower and essential oil. In this study, a member of the hydrolase gene family RrNUDX1 was cloned from Chinese traditional R. rugosa ‘Tang Hong’. Combined analysis of RrNUDX1 gene expression and the aroma components in different development stages and different parts of flower organ, we found that the main aroma component content was consistent with the gene expression pattern. The RrNUDX1 overexpressed Petunia hybrida was acquired via Agrobacterium-mediated genetic transformation systems. The blades of the transgenic petunias became wider and its growth vigor became strong with stronger fragrance. Gas chromatography with mass spectrometry analysis showed that the contents of the main aroma components of the transgenic petunias including methyl benzoate significantly increased. These findings indicate that the RrNUDX1 gene plays a role in enhancing the fragrance of petunia flowers, and they could lay an important foundation for the homeotic transformation of RrNUDX1 in R. rugosa for cultivating new R. rugosa varieties of high-yield and -quality essential oil.

An efficient transient transformation system for gene function studies in pumpkin (Cucurbita moschata D.)

Currently, gene exploration and molecular breeding in pumpkin (Cucurbita moschata Duch.) is a challenging process. There are limited efficient stable transformation methods for pumpkin. Transient transformation is a promising tool for the study of gene function. Here, an efficient Agrobacterium-mediated transient transformation system was developed for gene function studies in pumpkin. In this system, the function of salt-tolerant gene StNHX1 was confirmed by over-expression in germinated seeds. The CmPHD1-EGFP fusion protein was detected in the nucleus of cotyledon epidermal cells. This system can be used to analyze gene function and protein subcellular localization in pumpkin. Its advantages are highly efficient, cost-effective and time-saving (∼14 days). It may play a key role in gene exploration and molecular breeding in pumpkin, especially in large-scale analyses.

Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage, belonging to Brassica rapa species, is an important vegetable in Eastern Asia. It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low. The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies. In this study, we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors, with 93.50% regeneration rate on average. Based on this, a simple and efficient Agrobacterium-mediated genetic transformation method was established, without pre-culture procedure and concentration adjustment of hormone and AgNO3 in co-cultivation and selection media. Using this system, transformants could be obtained within 3.5–4.0 months. Average transformation frequency is up to 10.83%. The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

Developing a long-term and powerful in vitro culture and Agrobacterium-mediated transformation system for Miscanthus sinensis (Poaceae)

Miscanthus sinensis (Poaceae) is one of the perennial non-food biomass crops, whose germplasms have extensive use in Miscanthus breeding owing to its strong ecological adaptability and high level of genetic variation. However, the lack of a simple and powerful transformation system for this Miscanthus species remains a challenge for improving the trait performances and understanding their genetic mechanisms. In the present study, an extremely high percentage of embryogenic calli was obtained for different genotypes of M. sinensis using their mature seeds as the explants. Furthermore, the embryogenic calli were found to maintain a long-term high regeneration capacity (more than 2 years) when they were subcultured timely. The callus propagation, shoot regeneration, and root production of M. sinensis exhibited different tolerance ability to the selective agent including hygromycin, G418, and basta, and as a result, a novel selective culture strategy for identifying resistant transformants was developed to improve the efficiency of selection and transformation, in which the concentration of selective agent was decreased with different developmental stage. Optimizing the co-cultivation method and duration significantly increased the transformation frequency as well. Using the optimized protocol, a transformation frequency of 62.22 % or a positive transgenic plant frequency of 65.44 % was obtained for M. sinensis via Agrobacterium tumefaciens strain GV3101 harboring binary vector pCAMBIA2301. However, GUS enzyme activity in the transgenic lines of M. sinensis failed to be accurately detected by histochemical staining assay as well as by quantitative fluorescent assay, suggesting that GUS expression was disturbed in the transgenic plants and as a consequence, its results needed to be carefully interpreted. This study provided an efficient tool for cultivar improvement and genetic analysis of M. sinensis or other Miscanthus species.

A simple and fast Agrobacterium-mediated transformation system for passion fruit KPF4 (Passiflora edulis f. edulis\u2009\xd7\u2009Passiflora edulis f. flavicarpa)

BackgroundPassion fruit (Passiflora edulis Sims) is an important horticultural crop in the tropics and subtropics, where it has great commercial potential due to high demand for fresh edible fruits and processed juice as well as source of raw materials in cosmetic industries. Genetic engineering shows great potential in passion fruit improvement and can compensate for the limitations of conventional breeding. Despite the success achieved in genetic modification of few passion fruit varieties, transgenic passion fruit production is still difficult for farmer-preferred cultivars. Therefore, it is important to establish a simple and fast Agrobacterium-mediated cell transformation of commercial hybrid passion fruit KPF4 (Passiflora edulis f. edulis × Passiflora edulis f. flavicarpa).ResultsIn the present study, we have developed a simple and fast Agrobacterium-mediated transformation system for hybrid passion fruit KPF4 using leaf disc explants. Factors affecting the rate of transient beta (β)-glucuronidase (gusA) expression and consequently transformation efficiency were optimized as follows: Agrobacterium cell density with an OD600 of 0.5, 30 min infection time, 3 days of co-cultivation duration and the incorporation of 200 µM acetosyringone into Agrobacterium infection suspension medium. Using the optimized conditions, transgenic plants of KPF4 were produced within 2 months with an average transformation efficiency of 0.67%. The β-glucuronidase (GUS) histochemical staining confirmed the expression and integration of an intron-containing gusA gene into transformed leaf discs and transgenic plant lines of KPF4. The presence of gusA gene in the transgenic plants was confirmed by polymerase chain reaction (PCR). The results confirmed that the gusA gene was efficiently integrated into the passion fruit genome.ConclusionsThe developed transformation protocol is simple and rapid and could be useful for functional genomic studies and transferring agronomically important traits into passion fruit hybrid KPF4. This study developed a method that can be used to transfer traits such as resistance to viral diseases, low fruit quality and short storage life. To the best of our knowledge, this is the first report on genetic transformation system for commercial passion fruit hybrid KPF4.

An efficient genetic transformation system for Chinese medicine fungus Tolypocladium ophioglossoides

Tolypocladium ophioglossoides is a rare and valuable fungus extensively used in Chinese medicine for relieving postmenopausal syndrome in women yet its bioactive molecules are unknown. To explore its molecular mechanisms, we have developed a reliable Agrobacterium-mediated transformation system using the selective marker: the chlorimuron ethyl-resistance gene sur. For this purpose, we firstly constructed a T-DNA binary vector system and then improved the transformation efficiency by optimizing conditional parameters including the Agrobacterium tumefaciens concentration, the conidia number of T. ophioglossoides, the co-culture time and the concentration of acetosyringone. Furthermore, we have knocked-out the ku70 gene，which is a key gene in non-homologous end joining (NHEJ) DNA repair pathway，and the effect of the length of the homologous arms (HA) on the genetic transformation efficacy was also examined, which increased by 60% when HA was about 3 kb in length. Our results suggest that the genetic transformation system is efficient and feasible for the truffle-parasite fungus T. ophioglossoides, which can further be used in large-scale experiments for characterization of genes of interest in future work.

Efficient expressions of reporter genes in the industrial filamentous fungus Sclerotium rolfsii mediated by Agrobacterium tumefaciens

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters’ signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.

An efficient protocol for Agrobacterium-mediated genetic transformation of Antirrhinum majus

Snapdragon (Antirrhinum majus L.) is a popular ornamental and model plant species, and the recently released reference genome could greatly boost its utilization in fundamental research. However, the lack of an efficient genetic transformation system is still a major limiting factor for its full application in genetic and molecular studies. In this study, a simple method for quick regeneration and efficient Agrobacterium-mediated transformation of snapdragon was developed. Cotyledon petiole and hypocotyl explants derived from two-week-old seedlings were cultured on MS media supplemented with 2 mg/L zeatin (ZT), 0.2 mg/L 1-naphthaleneacetic acid (NAA), and 2 mg/L AgNO3, and adventitious shoots were regenerated through organogenesis with an average regeneration of 48.00% and 41.33%, respectively. By contrast, the regeneration frequency was only 22.67% for cotyledon petiole and 25.67% for hypocotyl explants in the absence of AgNO3. Moreover, the application of AgNO3 promoted indirect shoot organogenesis, while direct shoot organogenesis occurred in the absence of AgNO3 from both hypocotyl or cotyledon petiole explants. Agrobacterium-mediated genetic transformation systems were developed with this high-efficient regeneration system. The transformation efficiency has been improved from 0 to 1% through the direct shoot organogenesis to 3 to 4% via the indirect shoot organogenesis. This efficient regeneration and genetic transformation method could be important for future use of snapdragon as a model plant to address some fundamental questions which are hard to be solved by using other model plant species, and to accelerate the breeding process through CRISPR/Cas9 genome editing. Genetic transformation of snapdragon was significantly improved by applying AgNO3 to induce indirect organogenesis, which will substantially facilitate its fundamental research as well as molecular breeding.