Improving Sedum plumbizincicola genetic transformation with the SpGRF4-SpGIF1 gene and the self-excision CRE/LoxP system.

MAIN CONCLUSION: S. plumbizincicola genetic transformation was optimized using a self-excision molecular-assisted transformation system by integrating the SpGRF4/SpGIF1 gene with XVE and Cre/loxP. Sedum plumbizincicola, despite being an excellent hyperaccumulator of cadmium and zinc with significant potential for soil pollution phytoremediation on farmland, has nonetheless trailed behind other major model plants in genetic transformation technology. In this study, different explants and SpGRF4-SpGIF1 genes were used to optimize the genetic transformation of S. plumbizincicola. We found that petiole and stem segments had higher genetic transformation efficiency than cluster buds. Overexpression of SpGRF4-SpGIF1 could significantly improve the genetic transformation efficiency and shorten the period of obtaining regenerated buds. However, molecular assistance with overexpression of SpGRF4-SpGIF1 leads to abnormal morphology, resulting in plant tissue enlargement and abnormal growth. Therefore, we combined SpGRF4-SpGIF1 with XVE and Cre/loxP to obtain DNA autocleavage transgenic plants induced by estradiol, thereby ensuring normal growth in transgenic plants. This study optimized the S. plumbizincicola genetic transformation system, improved the efficiency of genetic transformation, and established a self-excision molecular-assisted transformation system. This work also established the basis for studying S. plumbizincicola gene function, and for S. plumbizincicola breeding and germplasm innovation.

In planta transformation in wheat: an improved protocol to develop wheat transformants.

BACKGROUND: Lack of efficient transformation protocol continues to be a major bottleneck for successful genome editing or transgenic development in wheat. An in planta transformation method was developed in Indian bread wheat in earlier study (Vasil et al. in Nat Biotechnol 10:667-674, 1992) which was labour-intensive and time-consuming. In the present study, in planta transformation method was improved to make it simple, efficient, less labour-intensive and time-saving. METHODS AND RESULTS: PCR-based screening for generated transformants at T0 stage was introduced in this method. Shoot apical meristem of two days old wheat seedling was inoculated with the routine active culture of Agrobacterium tumefaciens harboring plasmid pCAMBIA1300-Ubi-GFP having gene GFP under the control of Zea mays ubiquitin promoter. PCR analysis at T0 stage confirmed 27 plants to be transgene positive. These 27 plants were only taken to the next generation (T1) and the rest were discarded. At T1 generation 6 plants were analyzed to be PCR positive. Out of them, 4 plants were confirmed to have stable integration of transgene (GFP). Fluorescent microscopy at T1 stage confirmed the 4 Southern hybridization positive plants to be expressing reporter gene GFP. CONCLUSIONS: Screening at T0 stage, reduced the load of plants to be taken to T1 generation and their screening thereof at T1 with no overall loss in transformation efficiency. We successfully transformed wheat genotype HD2894 with 3.33% transformation efficiency using a simple, effective method which was less labour-intensive and less time-consuming. This method may be utilized to develop wheat transgenic as well as genome edited lines for desirable traits.

Development of a stable genetic transformation system in Erigeron breviscapus: a case study with EbYUC2 in relation to leaf number and flowering time.

MAIN CONCLUSION: A stable genetic transformation system for Erigeron breviscapus was developed. We cloned the EbYUC2 gene and genetically transformed it into Arabidopsis thaliana and E. breviscapus. The leaf number, YUC2 gene expression, and the endogenous auxin content in transgenic plants were significantly increased. Erigeron breviscapus is a prescription drug for the clinical treatment of cardiovascular and cerebrovascular diseases. The rosette leaves have the highest content of the major active compound scutellarin and are an important component in the yield of E. breviscapus. However, little is known about the genes related to the leaf number and flowering time of E. breviscapus. In our previous study, we identified three candidate genes related to the leaf number and flowering of E. breviscapus by combining resequencing data and genome-wide association study (GWAS). However, their specific functions remain to be characterized. In this study, we cloned and transformed the previously identified full-length EbYUC2 gene into Arabidopsis thaliana, developed the first stable genetic transformation system for E. breviscapus, and obtained the transgenic plants overexpressing EbYUC2. Compared with wild-type plants, the transgenic plants showed a significant increase in the number of leaves, which was correlated with the increased expression of EbYUC2. Consistently, the endogenous auxin content, particularly indole-3-acetic acid, in transgenic plants was also significantly increased. These results suggest that EbYUC2 may control the leaf number by regulating auxin biosynthesis, thereby laying a foundation for revealing the molecular mechanism governing the leaf number and flowering time of E. breviscapus.

Development of PEG-mediated genetic transformation and gene editing system of Bryum argenteum as an abiotic stress tolerance model plant.

KEY MESSAGE: To establish a sterile culture system and protoplast regeneration system for Bryum argenteum, and to establish and apply CRISPR/Cas9 system in Bryum argenteum. Bryum argenteum is a fascinating, cosmopolitan, and versatile moss species that thrives in various disturbed environments. Because of its comprehensive tolerance to the desiccation, high UV and extreme temperatures, it is emerging as a model moss for studying the molecular mechanisms underlying plant responses to abiotic stresses. However, the lack of basic tools such as gene transformation and targeted genome modification has hindered the understanding of the molecular mechanisms underlying the survival of B. argenteum in different environments. Here, we reported the protonema of B. argenteum can survive up to 95.4% water loss. In addition, the genome size of B. argenteum is approximately 313 Mb by kmer analysis, which is smaller than the previously reported 700 Mb. We also developed a simple method for protonema induction and an efficient protoplast isolation and regeneration protocol for B. argenteum. Furthermore, we established a PEG-mediated protoplast transient transfection and stable transformation system for B. argenteum. Two homologues of ABI3(ABA-INSENSITIVE 3) gene were successfully cloned from B. argenteum. To further investigate the function of the ABI3 gene in B. argenteum, we used the CRISPR/Cas9 genetic editing system to target the BaABI3A and BaABI3B gene in B. argenteum protoplasts. This resulted in mutagenesis at the target in about 2-5% of the regenerated plants. The isolated abi3a and abi3b mutants exhibited increased sensitivity to desiccation, suggesting that BaABI3A and BaABI3B play redundant roles in desiccation stress. Overall, our results provide a rapid and simple approach for molecular genetics in B. argenteum. This study contributes to a better understanding of the molecular mechanisms of plant adaptation to extreme environmental.

High-quality genome assembly and genetic transformation system of Lasiodiplodia theobromae strain LTTK16-3, a fungal pathogen of Chinese hickory.

Lasiodiplodia theobromae, as one of the causative agents associated with Chinese hickory trunk cankers, has caused huge economic losses to the Chinese hickory industry. Although the biological characteristics of this pathogen and the occurrence pattern of this disease have been well studied, few studies have addressed the related mechanisms due to the poor molecular and genetic study basis of this fungus. In this study, we sequenced and assembled L. theobromae strain LTTK16-3, isolated from a Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Phylogenetic analysis and comparative genomics analysis presented crucial cues in the prediction of LTTK16-3, which shared similar regulatory mechanisms of transcription, DNA replication, and DNA damage response with the other four Chinese hickory trunk canker-associated Botryosphaeria strains including, Botryosphaeria dothidea, Botryosphaeria fabicerciana, Botryosphaeria qingyuanensis, and Botryosphaeria corticis. Moreover, it contained 18 strain-specific protein clusters (not conserved in the other L. theobromae strains, AM2As and CITRA15), with potential roles in specific host-pathogen interactions during the Chinese hickory infection. Additionally, an efficient system for L. theobromae protoplast preparation and polyethylene glycol (PEG) -mediated genetic transformation was firstly established as the foundation for its future mechanisms study. Collectively, the high-quality genome data and the efficient transformation system of L. theobromae here set up the possibility of targeted molecular improvements for Chinese hickory canker control.IMPORTANCEFungi with disparate genomic features are physiologically diverse, possessing species-specific survival strategies and environmental adaptation mechanisms. The high-quality genome data and related molecular genetic studies are the basis for revealing the mechanisms behind the physiological traits that are responsible for their environmental fitness. In this study, we sequenced and assembled the LTTK16-3 strain, the genome of Lasiodiplodia theobromae first obtained from a diseased Chinese hickory tree (cultivar of Linan) in Linan, Zhejiang province, China. Further phylogenetic analysis and comparative genomics analysis provide crucial cues in the prediction of the proteins with potential roles in specific host-pathogen interactions during the Chinese hickory infection. An efficient PEG-mediated genetic transformation system of L. theobromae was established as the foundation for the future mechanisms exploration. The above genetic information and tools set up valuable clues to study L. theobromae pathogenesis and assist in Chinese hickory canker control.

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

Advances in the Transformation of Cyclamen persicum Mill. Through Direct Regeneration Based on an Optimized Kanamycin Selection Scheme.

Gene transfer technology has great value in ornamental plants toward the generation of varieties with new ornate characteristics. In the previous studies through the transformation of cyclamen, hygromycin was mainly used as a selective marker. However, there have been some drawbacks associated with hygromycin usage as a selecting agent. Therefore, in the current study, the optimization of kanamycin concentration in the regeneration media has been considered. Subsequently, the plant transformation using three different in vitro explants from three Cyclamen persicum cultivars using three Agrobacterium tumefaciens strains has been examined. Accordingly, the optimal kanamycin concentrations for regeneration from root and leaf explants were determined as 10 mg/L and for microtuber explants as 30 mg/L. The successful gene transformation in the antibiotic-resistant shoots were examined by PCR and UV-equipped microscopes. The gfp reporter gene transfer resulted in the highest efficiency of transformation (60%) to date, from the leaf explants of cv. Pure White inoculated with Agrobacterium tumefaciens strain LBA4404. In contrast, the lowest gene transfer efficiency (25%) was observed in root explants of cv. Dark Violet and cv. Neon Pink inoculated with strains GV3101 and AGL-1, respectively. The results of the current project are expandable to the subsequent investigations of Cyclamen persicum transformation.

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.

Establishment of an efficient callus transient transformation system for Vitis vinifera cv. 'Chardonnay'.

Grape (Vitis L.), a highly valued fruit crop, poses significant challenges in genetic transformation and functional characterization of genes. Therefore, there is an urgent need for the development of a rapid and effective method for grape transformation and gene function identification. Here, we introduce a streamlined Agrobacterium-mediated transient transformation system for grape calli. Optimal conditions were established with a leaf-derived callus induction medium; chiefly B5 medium supplemented with 0.05 mg/L NAA, 0.5 mg/L 2,4-D, and 2.0 mg/L KT; and a callus proliferation medium (B5 medium supplemented with 0.5 mg/L NAA and 2.0 mg/L 6-BA), respectively. Notably, GUS enzyme activity peaked (352.96 ± 33.95 mol 4-MU/mg/min) by sonication with Agrobacterium tumefaciens EHA105 and 100 µM AS for 4 min, followed by vacuum infection for 5 min, and co-culture at 25 °C in the dark for 1 day using callus as explants at an optical density (OD600) of 0.8. VaCIPK18 gene was transiently transformed into calli, and transcripts of the gene (endogenous and exogenous) were detected at higher levels than in non-transformed calli (endogenous). Moreover, after 10 days of treatment at 4 °C or -4 °C, the callus net weight of transformed callus was significantly higher than that of the untransformed callus, indicating that the VaCIPK18-overexpressing grape callus could improve cold tolerance. Overall, we establish a simple but effective transient transformation approach for grape callus, which could serve as a useful tool for the rapid assessment of gene function in this important crop.

A rapid and highly efficient sorghum transformation strategy using GRF4-GIF1/ternary vector system.

Sorghum is an important crop for food, forage, wine and biofuel production. To enhance its transformation efficiency without negative developmental by-effects, we investigated the impact of GRF4-GIF1 chimaera and GRF5 on sorghum transformation. Both GRF4-GIF1 and GRF5 effectively improved the transformation efficiency of sorghum and accelerated the transformation process of sorghum to less than 2 months which was not observed when using BBM-WUS. As agrobacterium  effectors increase the ability of T-DNA transfer into plant cells, we checked whether ternary vector system can additively enhance sorghum transformation. The combination of GRF4-GIF1 with helper plasmid pVS1-VIR2 achieved the highest transformation efficiency, reaching 38.28%, which is 7.71-fold of the original method. Compared with BBM-WUS, overexpressing GRF4-GIF1 caused no noticeable growth defects in sorghum. We further developed a sorghum CRISPR/Cas9 gene-editing tool based on this GRF4-GIF1/ternary vector system, which achieved an average gene mutation efficiency of 41.36%, and null mutants were created in the T0 generation.

An efficient targeted gene deletion approach for Cochliobolus heterostrophus using Agrobacterium tumefaciens-mediated transformation.

Cochliobolus heterostrophus is a plant pathogenic fungus of southern corn leaf blight, which has been regarded as a model necrotrophic plant pathogen. Many methods have been developed to knock out targeted genes in C. heterostrophus, of which the most widely-used one is protoplast-mediated transformation. However, there are several problems of this method associated with protoplast preparation, DNA product, time consumption, or high cost. In this study, a highly efficient target gene deletion approach in C. heterostrophus was established and optimized, based on Agrobacterium tumefaciens-mediated transformation (ATMT); the transformation efficiency of this approach was 85-88 transformants per 105 conidia, and the homologous recombination efficiency was approximately 68.3%. Furthermore, six gene knockout mutants of C. heterostrophus were obtained using this ATMT method. The phenotypes of this fungus altered in the mutant strains, and the virulence of the mutants significantly reduced compared to of the wild type strain. Taken together, this ATMT system established in this study can be used as a genetic manipulation tool for C. heterostrophus, to better understand the functions of genes and its relation to virulence.

Simple method for transformation and gene editing in medicinal plants.

A sample delivery method, modified from cut-dip-budding, uses explants with robust shoot regeneration ability, enabling transformation and gene editing in medicinal plants, bypassing tissue culture and hairy root formation. This method has potential for applications across a wide range of plant species.

Vacuum-Forced Agroinfiltration for In planta Transformation of Recalcitrant Plants: Cacao as a Case Study.

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.

A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes.

For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.

Genome-wide transformation reveals extensive exchange across closely related Bacillus species.

Bacterial transformation is an important mode of horizontal gene transfer that helps spread genetic material across species boundaries. Yet, the factors that pose barriers to genome-wide cross-species gene transfer are poorly characterized. Here, we develop a replacement accumulation assay to study the effects of genomic distance on transfer dynamics. Using Bacillus subtilis as recipient and various species of the genus Bacillus as donors, we find that the rate of orthologous replacement decreases exponentially with the divergence of their core genomes. We reveal that at least 96% of the B. subtilis core genes are accessible to replacement by alleles from Bacillus spizizenii. For the more distantly related Bacillus atrophaeus, gene replacement events cluster at genomic locations with high sequence identity and preferentially replace ribosomal genes. Orthologous replacement also creates mosaic patterns between donor and recipient genomes, rearranges the genome architecture, and governs gain and loss of accessory genes. We conclude that cross-species gene transfer is dominated by orthologous replacement of core genes which occurs nearly unrestricted between closely related species. At a lower rate, the exchange of accessory genes gives rise to more complex genome dynamics.

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Agrobacterium tumefaciens-mediated transformation of Nigrospora sp. isolated from switchgrass leaves and antagonistic toward plant pathogens.

Nigrospora is a diverse genus of fungi colonizing plants through endophytic, pathogenic, or saprobic interactions. Endophytic isolates can improve growth and development of host plants, as well as their resistance to microbial pathogens, but exactly how they do so remains poorly understood. Developing a reliable transformation method is crucial to investigate these mechanisms, in particular to identify pivotal genes for specific functions that correlate with specific traits. In this study, we identified eight isolates of Nigrospora sp. internally colonizing the leaves of switchgrass plants cultivated in North Carolina. Using an Agrobacterium tumefaciens-mediated transformation approach with control and GFP-expressing vectors, we report the first successful transformation of two Nigrospora isolates. Finally, we demonstrate that wild-type and transgenic isolates both negatively impact the growth of two plant pathogens in co-culture conditions, Bipolaris maydis and Parastagonospora nodorum, responsible for the Southern Leaf Blight and Septoria Nodorum Blotch diseases, respectively. The GFP-transformed strains developed here can therefore serve as accurate reporters of spatial interactions in future studies of Nigrospora and pathogens in the plant. Finally, the transformation method we describe lays the foundation for further genetic research on the Nigrospora genus to expand our mechanistic understanding of plant-endophyte interactions.

Establishment of a high-efficiency transformation and genome editing method for an essential vegetable and medicine Solanum nigrum.

Solanum nigrum, which belongs to the Solanaceae family, is an essential plant for food and medicine. It has many important secondary compounds, including glycoproteins, glycoalkaloids, polyphenolics, and anthocyanin-rich purple berries, as well as many ideal characteristics such as self-fertilization, a short life cycle and a small genome size that make it a potential model plant for the study of secondary metabolism and fruit development. In this study, we report a highly efficient and convenient tissue culture, transformation and genome editing method for S. nigrum using leaf segments after 8 weeks of tissue culture, with a required period from transformation initiation to harvest of about 3.5 months. Our results also show multi-shoot regeneration per leaf segment and a 100% shoot regeneration efficiency in a shoot regeneration medium. Moreover, over 82% of kanamycin-resistant plants exhibited strong green fluorescence marker protein expression, with genetic integration confirmed by PCR results and green fluorescence protein expression in their T1 progeny. Furthermore, we successfully applied this transformation method to achieve an average of 83% genome editing efficiency of SnMYB1, a gene involved in regulating the anthocyanin biosynthetic pathway of S. nigrum in response to missing nutrients. Taken together, the combination of highly efficient tissue culture, transformation and genome editing systems can provide a powerful platform for supporting fundamental research on the molecular mechanisms of secondary metabolism, fruit development, and production of important compounds by biotechnology.

Agrobacterium tumefaciens-mediated transformation of the white-rot fungus Dichomitus squalens.

Dichomitus squalens is an efficient white-rot fungus that generates a wide range of extracellular enzymes to degrade lignocellulose in nature. Although a protoplast-mediated transformation method for D. squalens has been developed, the transformation efficiency remains low. Here, we established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) procedure for D. squalens by transferring a binary vector harboring the neomycin phosphotransferase II (nptII) resistance gene fused with DsRed-Express2, under the control of the native glyceraldehyde-3-phosphate dehydrogenase (GPD) gene promoter. Key factors affecting the efficiency of transformation were tested. A. tumefaciens EHA105 strain with a cell density of 0.4 OD600nm and 96 h co-cultivation resulted in the highest transformation efficiency, with an average of 98 ± 11 transformants per co-cultivation plate. Besides, the strong expression of DsRed-Express2 indicates the effectiveness of the DsGPD promoter in driving gene expression in D. squalens. This ATMT system of D. squalens would be beneficial for its molecular genetic studies.

Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis.

Citrus is one of the major horticultural crops with high economic and nutraceutical value. Despite the fact that conventional research has developed numerous improved varieties, citriculture is still susceptible to various stresses and requires innovative solutions such as genetic engineering. Among all the currently available modern approaches, Agrobacterium-mediated transformation is the most efficient method for introducing desired traits in citrus. However, being a non-host for Agrobacterium, various citrus species, including Citrus aurantifolia and Citrus sinensis, are recalcitrant to this method. The available reports on Agrobacterium-mediated transformation of commercial citrus cultivars show very low transformation efficiency with poor recovery rates of whole transgenic plantlets. Here, we provide an efficient and reliable procedure of Agrobacterium-mediated transformation for both C. aurantifolia and C. sinensis. This protocol depends on providing callus-inducing treatment to explants before and during Agrobacterium co-cultivation, using optimum conditions for shoot regeneration and modifying in-vitro micrografting protocol to combat the loss of transgenic lines. As transgenic citrus shoots are difficult to root, we also developed the ideal conditions for their rooting. Using this protocol, the whole transgenic plantlets of C. aurantifolia and C. sinensis can be developed in about ~ 4 months, with transformation efficiency of 30% and 22% for the respective species.