Robust soybean leaf agroinfiltration.

KEY MESSAGE: A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and Agrobacterium culture conditions are important for successful agroinfiltration. Agroinfiltration of Nicotiana benthamiana has emerged as a workhorse transient assay for plant biotechnology and synthetic biology to test the performance of gene constructs in dicot leaves. While effective, it is nonetheless often desirable to assay transgene constructs directly in crop species. To that end, we innovated a substantially robust agroinfiltration method for Glycine max (soybean), the most widely grown dicot crop plant in the world. Several factors were found to be relevant to successful soybean leaf agroinfiltration, including genotype, surfactant, developmental stage, and Agrobacterium strain and culture medium. Our optimized protocol involved a multi-step Agrobacterium culturing process with appropriate expression vectors, Silwet L-77 as the surfactant, selection of fully expanded leaves in the VC or V1 stage of growth, and 5 min of vacuum at - 85 kPa followed by a dark incubation period before plants were returned to normal growth conditions. Using this method, young soybean leaves of two lines-V17-0799DT, and TN16-5004-were high expressors for GUS, two co-expressed fluorescent protein genes, and the RUBY reporter product, betalain. This work not only represents a new research tool for soybean biotechnology, but also indicates critical parameters for guiding agroinfiltration optimization for other crop species. We speculate that leaf developmental stage might be the most critical factor for successful agroinfiltration.

Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update.

In this protocol, we outline how to produce a chimeric viral vaccine in a biosafety level 1 (BSL1) environment. An animal viral vector RNA encapsidated with tobacco mosaic virus (TMV) coat protein can be fully assembled in planta. Agrobacterium cultures containing each component are inoculated together into tobacco leaves and the self-assembled hybrid chimeric viral vaccine is harvested 4 days later and purified with a simple PEG precipitation. The viral RNA delivery vector is derived from the BSL1 insect virus, Flock House virus (FHV), and replicates in human and animal cells but does not spread systemically. A polyethylene glycol purification protocol is also provided to collect and purify these vaccines for immunological tests. In this update, we also provide a protocol for in trans co-inoculation of a modified FHV protein A, which significantly increased the yield of in planta chimeric viral vaccine.

Adding value to banana farming: Antibody production in post-harvest leaves.

Banana, a globally popular fruit, is widely cultivated in tropical and sub-tropical regions. After fruit harvest, remaining banana plant materials are low-value byproducts, mostly composted or used as fibre or for food packaging. As an aim to potentially increase farmer income, this study explored underutilised banana biomass as a novel plant tissue for production of a high-value product. Protein scFvTG130 used in this study, is an anti-toxoplasma single chain variable fragment antibody that can be used in diagnostics and neutralising the Toxoplasma gondii pathogen. Using detached banana leaves, we investigated the factors influencing the efficacy of a transient expression system using reporter genes and recombinant protein, scFvTG130. Transient expression was optimal at 2 days after detached banana leaves were vacuum infiltrated at 0.08 MPa vacuum pressure for a duration of 3 min with 0.01% (v/v) Tween20 using Agrobacterium strain GV3101 harbouring disarmed virus-based vector pIR-GFPscFvTG130. The highest concentration of anti-toxoplasma scFvTG130 antibody obtained using detached banana leaves was 22.8 µg/g fresh leaf tissue. This first study using detached banana leaf tissue for the transient expression of a recombinant protein, successfully demonstrated anti-toxoplasma scFvTG130 antibody expression, supporting the potential application for other related proteins using an underutilised detached banana leaf tissue.

Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

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.

Withania somnifera (L.) Dunal: Enhanced production of withanolides and phenolic acids from hairy root culture after application of elicitors.

Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14 mg l-1) induced 11.49 -fold increase in withanolides (89.07 ± 2.75 mg g-1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11 mg g- 1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63 mg g-1 DW of withanolides and 15.66 ± 0.92 mg g-1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids.

Compartmentalized Terpenoid Production in Plants Using Agrobacterium-Mediated Transient Expression.

As the field of plant synthetic biology continues to grow, Agrobacterium-mediated transient expression has become an essential method to rapidly test pathway candidate genes in a combinatorial fashion. This is especially important when elucidating and engineering more complex pathways to produce commercially relevant chemicals like many terpenoids, a widely diverse class of natural products of often industrial relevance. Agrobacterium-mediated transient expression has facilitated multiplex expression of recombinant and modified enzymes, including synthetic biology approaches to compartmentalize the biosynthesis of terpenoids subcellularly. Here, we describe methods on how to deploy Agrobacterium-mediated transient expression in Nicotiana benthamiana to rapidly develop terpenoid pathways and compartmentalize terpenoid biosynthesis within plastids, the cytosol, or at the surface of lipid droplets.

Generation of CRISPR-edited birch plants without DNA integration using Agrobacterium-mediated transformation technology.

CRISPR/Cas9 system has emerged as a powerful tool in genome editing; however, generation of CRISPR-edited DNA-free plants is still challenging. In this study, Betula platyphylla (birch) was used to build a method to generate CRISPR-edited plant without foreign DNA integration using Agrobacterium-mediated transformation (CPDAT method). This technique utilizes transient genetic transformation to introduce T-DNA coding gRNA and Cas9 into birch cells, and T-DNA will express to synthesize gRNA and Cas9 protein, which will form a complex to cleave the target DNA site. The genome may be mutated due to DNA repair, and these mutations will be preserved and accumulated not dependent on whether T-DNA is integrated into the genome or not. After transient transformation, birch plants were cut into explants to induce adventitious buds without antibiotic selection pressure. Each adventitious bud can be considered as an independent potentially CRISPR-edited line for mutation detection. CRISPR-edited birch plants without foreign DNA integration are further selected by screening CRISPR-edited lines without T-DNA integration. Among 65 randomly chosen independent lines, the mutation rate was 80.00% including 40.00% of lines with both alleles mutated. In addition, 5 lines out of 65 studied lines (7.69%) were CRISPR-edited birch plants without DNA integration. In conclusion, this innovative method presents a novel strategy for generating CRISPR-edited birch plants, thereby significantly enhancing the efficiency of generating common CRISPR-edited plants. These findings offer considerable potential to develop plant genome editing techniques further.

Intragenic Agrobacterium-mediated gene transfer mimics micro-translocations without foreign DNA.

MAIN CONCLUSION: Agrobacterium-mediated transformation of Nicotiana tabacum, using an intragenic T-DNA region derived entirely from the N. tabacum genome, results in the equivalence of micro-translocations within genomes. Intragenic Agrobacterium-mediated gene transfer was achieved in Nicotiana tabacum using a T-DNA composed entirely of N. tabacum DNA, including T-DNA borders and the acetohydroxyacid synthase gene conferring resistance to sulfonylurea herbicides. Genomic analysis of a resulting plant, with single locus inheritance of herbicide resistance, identified a single insertion of the intragenic T-DNA on chromosome 5. The insertion event was composed of three N. tabacum DNA fragments from other chromosomes, as assembled on the T-DNA vector. This validates that intragenic transformation of plants can mimic micro-translocations within genomes, with the absence of foreign DNA.

Cloning of maize chitinase 1 gene and its expression in genetically transformed rice to confer resistance against rice blast caused by Pyricularia oryzae.

Fungal pathogens are one of the major reasons for biotic stress on rice (Oryza sativa L.), causing severe productivity losses every year. Breeding for host resistance is a mainstay of rice disease management, but conventional development of commercial resistant varieties is often slow. In contrast, the development of disease resistance by targeted genome manipulation has the potential to deliver resistant varieties more rapidly. The present study reports the first cloning of a synthetic maize chitinase 1 gene and its insertion in rice cv. (Basmati 385) via Agrobacterium-mediated transformation to confer resistance to the rice blast pathogen, Pyricularia oryzae. Several factors for transformation were optimized; we found that 4-week-old calli and an infection time of 15 minutes with Agrobacterium before colonization on co-cultivation media were the best-suited conditions. Moreover, 300 µM of acetosyringone in co-cultivation media for two days was exceptional in achieving the highest callus transformation frequency. Transgenic lines were analyzed using molecular and functional techniques. Successful integration of the gene into rice lines was confirmed by polymerase chain reaction with primer sets specific to chitinase and hpt genes. Furthermore, real-time PCR analysis of transformants indicated a strong association between transgene expression and elevated levels of resistance to rice blast. Functional validation of the integrated gene was performed by a detached leaf bioassay, which validated the efficacy of chitinase-mediated resistance in all transgenic Basmati 385 plants with variable levels of enhanced resistance against the P. oryzae. We concluded that overexpression of the maize chitinase 1 gene in Basmati 385 improved resistance against the pathogen. These findings will add new options to resistant germplasm resources for disease resistance breeding. The maize chitinase 1 gene demonstrated potential for genetic improvement of rice varieties against biotic stresses in future transformation programs.

[High-level expression of anti FLAG tag antibody in plants].

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.

Protocol for imbibed seed piercing for Agrobacterium-mediated transformation of jute.

Here, we present a streamlined Agrobacterium-mediated transformation protocol for jute (Corchorus sp.). We describe steps to pierce and vacuum infiltrate imbibed jute seeds with Agrobacterium suspension. We then detail procedures for selecting transformed seeds by using a hygromycin-B-supplemented medium. This approach can achieve transformation efficiencies of 20.44% ± 1.17% and 15.55% ± 0.58% for tossa (C. olitorius) and white (C. capsularis) jute, respectively. Demanding minimal resources and time, this protocol can elevate genetic engineering research in jute fiber crops. For complete details on the use and execution of this protocol, please refer to Majumder et al. (2020).1.

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.

Revealing the phosphate-solubilizing characteristics and mechanisms of the plant growth-promoting bacterium Agrobacterium deltaense C1.

AIMS: This study explores the phosphate (Pi)-solubilizing characteristics and mechanisms of a novel phosphate-solubilizing bacterium, Agrobacterium deltaense C1 (C1 hereafter). METHODS AND RESULTS: The growth-promoting effects of C1 were investigated by gnotobiotic experiments, and the Pi-solubilizing mechanism was revealed by extracellular metabolomics, liquid chromatography analysis, and reverse transcription quantitative polymerase chain reaction. Results showed that C1 significantly increased Arabidopsis biomass and total phosphorus (P) content under P deficiency. Under Ca3(PO4)2 condition, the presence of C1 resulted in a significant and negative correlation between available P content and medium pH changes, implying that Pi dissolution occurs through acid release. Metabolomics revealed C1's ability to release 99 organic acids, with gluconic acid (GA), citric acid, and α-ketoglutaric acid contributing 64.86%, 9.58%, and 0.94%, respectively, to Pi solubilization. These acids were significantly induced by P deficiency. Moreover, C1's Pi solubilization may remain significant even in the presence of available P, as evidenced by substantial pH reduction and high gcd gene expression. Additionally, C1 produced over 10 plant growth-promoting substances. CONCLUSIONS: C1 dissolves Pi primarily by releasing GA, which enhances plant growth under P deficiency. Notably, its Pi solubilization effect is not significantly limited by available Pi.

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.

Extensive natural Agrobacterium-induced transformation in the genus Camellia.

MAIN CONCLUSION: The genus Camellia underwent extensive natural transformation by Agrobacterium. Over a period of 15 million years, at least 12 different inserts accumulated in 72 investigated Camellia species. Like a wide variety of other wild and cultivated plants, Camellia species carry cellular T-DNA sequences (cT-DNAs) in their nuclear genomes, resulting from natural Agrobacterium-mediated transformation. Short and long DNA sequencing reads of 435 accessions belonging to 72 Camellia species (representing 12 out of 14 sections) were investigated for the occurrence of cT-DNA insertions. In all, 12 different cT-DNAs were recovered, either completely or partially, called CaTA to CaTL. Divergence analysis of internal cT-DNA repeats revealed that the insertion events span a period from 0.075 to 15 Mio years ago, and yielded an average transformation frequency of one event per 1.25 Mio years. The two oldest inserts, CaTA and CaTD, have been modified by spontaneous deletions and inversions, and by insertion of various plant sequences. In those cases where enough accessions were available (C. japonica, C. oleifera, C. chekiangoleosa, C. sasanqua and C. pitardii), the younger cT-DNA inserts showed a patchy distribution among different accessions of each species, indicating that they are not genetically fixed. It could be shown that Camellia breeding has led to intersectional transfer of cT-DNAs. Altogether, the cT-DNAs cover 374 kb, and carry 47 open reading frames (ORFs). Two Camellia cT-DNA genes, CaTH-orf358 and CaTK-orf8, represent new types of T-DNA genes. With its large number of cT-DNA sequences, the genus Camellia constitutes an interesting model for the study of natural Agrobacterium transformants.

Efficient CRISPR/Cas9-mediated genome editing in sheepgrass (Leymus chinensis).

The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing in a perennial, stress-tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single-guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co-expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass.

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.

Wheat gibberellin oxidase genes and their functions in regulating tillering.

Multiple genetic factors control tillering, a key agronomy trait for wheat (Triticum aestivum L.) yield. Previously, we reported a dwarf-monoculm mutant (dmc) derived from wheat cultivar Guomai 301, and found that the contents of gibberellic acid 3 (GA3) in the tiller primordia of dmc were significantly higher. Transcriptome analysis indicated that some wheat gibberellin oxidase (TaGAox) genes TaGA20ox-A2, TaGA20ox-B2, TaGA3ox-A2, TaGA20ox-A4, TaGA2ox-A10 and TaGA2ox-B10 were differentially expressed in dmc. Therefore, this study systematically analyzed the roles of gibberellin oxidase genes during wheat tillering. A total of 63 TaGAox genes were identified by whole genome analysis. The TaGAoxs were clustered to four subfamilies, GA20oxs, GA2oxs, GA3oxs and GA7oxs, including seven subgroups based on their protein structures. The promoter regions of TaGAox genes contain a large number of cis-acting elements closely related to hormone, plant growth and development, light, and abiotic stress responses. Segmental duplication events played a major role in TaGAoxs expansion. Compared to Arabidopsis, the gene collinearity degrees of the GAoxs were significantly higher among wheat, rice and maize. TaGAox genes showed tissue-specific expression patterns. The expressions of TaGAox genes (TaGA20ox-B2, TaGA7ox-A1, TaGA2ox10 and TaGA3ox-A2) were significantly affected by exogenous GA3 applications, which also significantly promoted tillering of Guomai 301, but didn't promote dmc. TaGA7ox-A1 overexpression transgenic wheat lines were obtained by Agrobacterium mediated transformation. Genomic PCR and first-generation sequencing demonstrated that the gene was integrated into the wheat genome. Association analysis of TaGA7ox-A1 expression level and tiller number per plant demonstrated that the tillering capacities of some TaGA7ox-A1 transgenic lines were increased. These data demonstrated that some TaGAoxs as well as GA signaling were involved in regulating wheat tillering, but the GA signaling pathway was disturbed in dmc. This study provided valuable clues for functional characterization of GAox genes in wheat.