Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.

Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 µM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 µM) and BAP (2,2 µM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.

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.

Agroinfiltration for Enhanced Transgene Expression in Coffee Leaves (Coffea arabica L.).

The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

CRISPR/Cas9 Vector Construction for Gene Knockout.

This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352 bp) and scaffold (83 bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide. The three gRNA expression fragments are then loosely assembled in a ligation-free cloning reaction and used as a template for an additional PCR step to amplify a single gRNA expression construct, ready for assembly into the transformation vector. The benefits of this design include cost efficiency, as subsequent cloning reactions only require 59 oligonucleotides and standard cloning reagents. Researchers engaged in CRISPR/Cas9-mediated genome editing in plants will find this protocol a clear and resource-efficient approach to create transformation plasmids for their experiments.

Genome Editing in Brassica juncea Using CRISPR/Cas9 Technology.

Modern genome editing tools particularly CRISPR/Cas9 have revolutionized plant genome manipulation for engineering resilience against changing climatic conditions, disease infestation, as well as functional genomic studies. CRISPR-mediated genome editing allows for editing at a single as well as multiple locations in the genome simultaneously, making it an effective tool for polyploid species too. However, still, its applications are limited to the model crops only. Extending it to crop plants will help improve field crops against the changing climates more rapidly and precisely. Here we describe the protocol for editing the genome of a field crop Brassica juncea (mustard), an allotetraploid and important oilseed crop of the Indo-Pak Subcontinent region. This protocol is based on the Agrobacterium-mediated transformation for the delivery of CRISPR components into the plant genome using cotyledon as explants. We elaborate on steps for recovering genome-edited knockouts, for validation of the edits, as well as recovering the transgene-free edited plants through a commonly used segregating approach.

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.

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 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.