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.

GATA transcription factor in common bean: A comprehensive genome-wide functional characterization, identification, and abiotic stress response evaluation.

The GATA transcription factors (TFs) have been extensively studied for its regulatory role in various biological processes in many plant species. The functional and molecular mechanism of GATA TFs in regulating tolerance to abiotic stress has not yet been studied in the common bean. This study analyzed the functional identity of the GATA gene family in the P. vulgaris genome under different abiotic and phytohormonal stress. The GATA gene family was systematically investigated in the P. vulgaris genome, and 31 PvGATA TFs were identified. The study found that 18 out of 31 PvGATA genes had undergone duplication events, emphasizing the role of gene duplication in GATA gene expansion. All the PvGATA genes were classified into four significant subfamilies, with 8, 3, 6, and 13 members in each subfamily (subfamilies I, II, III, and IV), respectively. All PvGATA protein sequences contained a single GATA domain, but subfamily II members had additional domains such as CCT and tify. A total of 799 promoter cis-regulatory elements (CREs) were predicted in the PvGATAs. Additionally, we used qRT-PCR to investigate the expression profiles of five PvGATA genes in the common bean roots under abiotic conditions. The results suggest that PvGATA01/10/25/28 may play crucial roles in regulating plant resistance against salt and drought stress and may be involved in phytohormone-mediated stress signaling pathways. PvGATA28 was selected for overexpression and cloned into N. benthamiana using Agrobacterium-mediated transformation. Transgenic lines were subjected to abiotic stress, and results showed a significant tolerance of transgenic lines to stress conditions compared to wild-type counterparts. The seed germination assay suggested an extended dormancy of transgenic lines compared to wild-type lines. This study provides a comprehensive analysis of the PvGATA gene family, which can serve as a foundation for future research on the function of GATA TFs in abiotic stress tolerance in common bean plants.

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.

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.

Melatonin supplementation enhances browning suppression and improves transformation efficiency and regeneration of transgenic rough lemon plants (Citrus × jambhiri).

Enzymatic browning poses a significant challenge that limits in vitro propagation and genetic transformation of plant tissues. This research focuses on investigating how adding antioxidant substances can suppress browning, leading to improved efficiency in transforming plant tissues using Agrobacterium and subsequent plant regeneration from rough lemon (Citrus × jambhiri). When epicotyl segments of rough lemon were exposed to Agrobacterium, they displayed excessive browning and tissue decay. This was notably different from the 'Hamlin' explants, which did not exhibit the same issue. The regeneration process failed completely in rough lemon explants, and they accumulated high levels of total phenolic compounds (TPC) and polyphenol oxidase (PPO), which contribute to browning. To overcome these challenges, several antioxidant and osmoprotectant compounds, including lipoic acid, melatonin, glycine betaine, and proline were added to the tissue culture medium to reduce the oxidation of phenolic compounds and mitigate browning. Treating epicotyl segments with 100 or 200 µM melatonin led to a significant reduction in browning and phenolic compound accumulation. This resulted in enhanced shoot regeneration, increased transformation efficiency, and reduced tissue decay. Importantly, melatonin supplementation effectively lowered the levels of TPC and PPO in the cultured explants. Molecular and physiological analyses also confirmed the successful overexpression of the CcNHX1 transcription factor, which plays a key role in imparting tolerance to salinity stress. This study emphasizes the noteworthy impact of supplementing antioxidants in achieving successful genetic transformation and plant regeneration in rough lemon. These findings provide valuable insights for developing strategies to address enzymatic browning and enhance the effectiveness of plant tissue culture and genetic engineering methods with potential applications across diverse plant species.

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.

Cloning and functional analysis of ZmMADS42 gene in maize.

Maize (Zea mays L.) is the most important cereal crop in the world. Flowering period and photoperiod play important roles in the reproductive development of maize. This study, investigated ZmMADS42, a gene that is highly expressed in the shoot apical meristem. Agrobacterium infection was used to successfully obtain overexpressed ZmMADS42 plants. Fluorescence quantitative PCR revealed that the expression of the ZmMADS42 gene in the shoot apical meristem of transgenic plants was 2.8 times higher than that of the wild-type(WT). In addition, the expression of the ZmMADS42 gene in the endosperm was 2.4 times higher than that in the wild-type. The seed width of the T2 generation increased by 5.35%, whereas the seed length decreased by 7.78% compared with that of the wild-type. Dissection of the shoot tips of transgenic and wild-type plants from the 7-leaf stage to the 9-leaf stage revealed that the transgenic plants entered the differentiation stage earlier and exhibited more tassel meristems during their vegetative growth period. The mature transgenic plants were approximately 20 cm shorter in height and had a lower panicle position than the wild-type plants. Comparing the flowering period, the tasseling, powdering, and silking stages of the transgenic plants occurred 10 days earlier than those of the wild-type plants. The results showed that the ZmMADS42 gene played a significant role in regulating the flowering period and plant height of maize.

Ether Bond Cleavage of a Phenylcoumaran ß-5 Lignin Model Compound and Polymeric Lignin Catalysed by a LigE-type Etherase from Agrobacterium sp.

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a ß-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C-C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin.

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.

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.

The Targeted Regulation of BDUbc and BDSKL1 Enhances Resistance to Blight in Bambusa pervariabilis × Dendrocalamopsis grandis.

Arthrinium phaeospermum is the major pathogen responsible for the significant stem disease "blight" in B. pervariabilis × D. grandis. The interacting proteins of the key pathogenic factor ApCtf1ß, BDUbc and BDSKL1, have previously been obtained by two-hybrid, BiFC, GST pull-down yeast assays. However, the functions of these interacting proteins remain unknown. This study successfully obtained transgenic plants overexpressing BDUbc, BDSKL1, and BDUbc + BDSKL1 via Agrobacterium-mediated gene overexpression. qRT-PCR analysis revealed significantly increased expression levels of BDUbc and BDSKL1 in the transgenic plants. After infection with the pathogenic spore suspension, the disease incidence and severity index significantly decreased across all three transgenic plants, accompanied by a marked increase in defense enzyme levels. Notably, the co-transformed plant, OE-BDUbc + BDSKL1, demonstrated the lowest disease incidence and severity index among the transgenic variants. These results not only indicate that BDUbc and BDSKL1 are disease-resistant genes, but also that these two genes may exhibit a synergistic enhancement effect, which further improves the resistance to blight in Bambusa pervariabilis × Dendrocalamopsis grandis.

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.

Genetic manipulation of Indian mustard genotypes with WRR-gene(s) confers resistance against Albugo candida.

BACKGROUND: Brassica species is the second most important edible oilseed crop in India. Albugo candida (Pers.) Kuntze, a major oomycete disease of oilseed brassica causing white rust, leads to 60% yield loss globally. The prevalence of A. candida race 2 (Ac2V) that specifically infects B. juncea, coupled with limitations of conventional methods has resulted in a dearth of white rust resistance resources in cultivated varieties. METHODS AND RESULTS: In an effort to develop resistant plants, Agrobacterium mediated genetic transformation of three B. juncea genotypes viz., susceptible host var. Varuna, along with its doubled haploid mutant lines C66 and C69 (showing moderate tolerance to field isolates of A. candida) was initiated to transfer resistance genes (WRR8Sf-2 and WRR9Hi-0) identified in Arabidopsis thaliana against race Ac2V, that encode for Toll-like/interleukin-1 receptor-nucleotide binding-leucine-rich repeat proteins that recognize effectors of the pathogen races. CONCLUSIONS: Our results demonstrate that introduction of resistance genes from a tertiary gene pool by genetic transformation enhances disease resistance in B. juncea genotypes to a highly virulent Ac2V isolate.

[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.

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.

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.

High-level expression of anti FLAG tag antibody in plants / 生物工程学报

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.