It is crucial that experiments dealing with plant–pathogen interactions are robust and reproducible. For evaluating the disease‐resistance ability of a host plant against any pathogen, controlled conditions are a prerequisite that cannot be obtained with soil‐based systems. Variation in environmental conditions during challenge inoculation of a pathogen, when performed in fields or a polyhouse, may lead to spurious results. This is of particular importance when dealing with disease resistance in a fruit tree like pomegranate, as determined after years of research efforts. Due to lack of uniformity and environment variability, the possibility of pathogen escapes and false positives is large under field or polyhouse conditions. To address these limitations, we have used tissue‐cultured plants and developed an easy, reliable, rapid, and efficient in vitro bioassay for bacterial blight on pomegranate. Owing to its performance under a closed system, this system minimizes the risk of pathogen escapes, reduces environmental variability, and provides a robust high‐throughput screening platform for disease resistance and tissue sampling for downstream functional genomics studies. The protocols enable reproducible experiments to study plant response to bacterial blight infection. Stepwise protocols and detailed instructions are provided to carry out the screening experiment in pomegranate or other similar plant species. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Preparation of nodal explants and in vitro rooting Basic Protocol 2 : In vitro blight bioassay Basic Protocol 3 : Pathogen re‐isolation and confirmation
Lathyrus sativus, commonly known as the grass pea, is a nutritious legume that is resilient to climate change, allowing it to grow in drought, waterlogged, and saline soils. However, developing effective functional genomic tools for this crop has been challenging, primarily due to the absence of reliable and stable transformation protocols. Agrobacterium rhizogenes-mediated hairy root transformation provides a practical and rapid method for validating gene functions using the CRISPR/Cas system. This method has not been applied to grass pea despite its potential. In this article, we present the first protocol for A. rhizogenes-mediated hairy root transformation and CRISPR/Cas genome editing aimed at the functional characterization of candidate genes in L. sativus. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Designing CRISPR/Cas9 construct for targeted gene editing in L. sativus Support Protocol 1: Escherichia coli competent cell preparation and transformation Support Protocol 2: A. rhizogenes competent cell preparation and transformation Basic Protocol 2: A. rhizogenes-mediated hairy root transformation in L. sativus Basic Protocol 3: Screening of transgenic hairy root lines Support protocol 3: DNA isolation from L. sativus hairy roots.
This set of protocols was developed to detect Ascochyta rabiei, the fungus responsible for chickpea blight, in seeds. Given the significant impact of this disease and its potential to cause complete crop loss, even at low inoculum levels, optimizing detection protocols is crucial. This article proposes adjustments to key variables of a basic agar plate test-disinfection method, incubation, illumination, and culture medium-to improve microscopic identification (mainly using a stereomicroscope) and enhance differential growth of A. rabiei relative to other seed-borne fungi. Each step was carefully validated. Detection was verified through morphological and molecular confirmation of A. rabiei, as well as interlaboratory repeatability tests. In addition, the first distribution map of A. rabiei in Argentina was generated based on chickpea seed analyses conducted in the past decade and using the protocols outlined in this article. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Seed sampling procedure Basic Protocol 2: Preparation of culture medium Support Protocol: Sterilization of antibiotic Basic Protocol 3: Seed disinfection by washing Basic Protocol 4: Seed sowing and incubation Basic Protocol 5: Detection of infected seeds.
High-precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated 9) system is a programmable, RNA-guided nuclease that introduces targeted, site-specific double-stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame-shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step-by-step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CRISPR/Cas9 guide RNA target selection Support Protocol 1: Genomic DNA extraction in-house protocol Basic Protocol 2: Construction of a binary plasmid vector Support Protocol 2: Agrobacterium transformation with a binary vector construct and stability check Support Protocol 3: Plant transformation Basic Protocol 3: Genotyping of edited events.
RNA G-quadruplexes (RG4s) are specific and complex tertiary structures that form in guanine-rich regions of RNA and can be detected in vitro. A recently developed transcriptome-wide technique, SHALiPE-Seq, enables the assessment of RG4 folding status within living cells in a quantitative manner. This method integrates chemical probing with high-throughput sequencing. SHALiPE-Seq relies on the property of 2-methylnicotinic acid imidazolide (NAI), which preferentially modifies the last guanine in G-tracts when RG4s are folded. To establish reference profiles, in vitro NAI modification patterns are generated under potassium ion (K⁺) conditions, which promote folding, and lithium ion (Li⁺) conditions, which maintain RG4s in an unfolded state. By comparing in vivo SHALiPE-Seq profiles with these in vitro benchmarks, it becomes possible to identify and evaluate the formation of RG4s in living cells. Although this protocol has been applied to Arabidopsis thaliana and rice, SHALiPE-Seq is broadly applicable to other systems and provides a valuable approach for investigating the in vivo dynamics of RG4s and their potential biological functions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: In vitro and in vivo NAI probing of RNA Basic Protocol 2: Construction of the SHALiPE-Seq libraries Basic Protocol 3: Measurement of the RNA G-quadruplex folding status based on SHALiPE-Seq libraries.
