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.
Milkweeds have ecological significance for insect herbivores that rely on them as hosts for either part of or the entirety of their life cycles. Interesting interactions, some of which are not completely understood, have evolved over time. To develop these species as models to elucidate the interplay with insect herbivores, we established Agrobacterium tumefaciens-mediated transformation approaches for Asclepias hallii (Hall's milkweed), A. syriaca (common milkweed), and A. tuberosa (butterflyweed). The method is based on infection of stem internodal explants, which were more amenable to transformation than leaf explants. We found that addition of freshly prepared dithiothreitol was critical to prevent browning of stem explants. Depending on the species, the time from infection to the regeneration of transgenic lines ranges from 2 to 4 months. Transformation efficiency for A. hallii was 9%, whereas efficiencies for A. syriaca and A. tuberosa were 6% and 13%, respectively. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Agrobacterium tumefaciens-mediated transformation of Asclepias internodal stem explants Basic Protocol 2: Preparation of Agrobacterium glycerol stocks containing gene constructs.
Isolation of high-quality DNA from infected plant specimens is an essential step for the molecular detection of plant pathogens. However, DNA isolation from plant cells surrounded by rigid polysaccharide cell walls involves complicated steps and requires benchtop laboratory equipment. As a result, plant DNA extraction is currently confined to well-equipped laboratories and sample preparation has become one of the major hurdles for on-site molecular detection of plant pathogens. To overcome this hurdle, a simple DNA extraction method from plant leaf tissues has been developed. A microneedle (MN) patch made of polyvinyl alcohol (PVA) can isolate plant or pathogenic DNA from different plant species within a minute. During DNA extraction, the polymeric MN patch penetrates into plant leaf tissues and breaks rigid plant cell walls to isolate intracellular DNA. The extracted DNA is polymerase chain reaction (PCR) amplifiable without additional purification. This minimally invasive method has successfully extracted Phytophthora infestans DNA from infected tomato leaves. Moreover, the MN patch could be used to isolate DNA from other plant pathogens directly in the field. Thus, it has great potential to become a rapid, on-site sample preparation technique for plant pathogen detection. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Microneedle patch-based DNA extraction Support Protocol 1: Microneedle patch fabrication Support Protocol 2: Real-time PCR amplification of microneedle patch extracted DNA.
The ability to sequence DNA retrieved from ancient and historical material plays a crucial role in reinforcing evolutionary and anthropological inference. While the focus of the field is largely on analyzing DNA from ancient hominids and other animals, we have also learned from plant ancient DNA (aDNA), in particular, about human farming practices, crop domestication, environment management, species invasion, and adaptation to various environmental conditions. In the following protocols, we outline best practices for plant aDNA isolation, preparation for sequencing, bioinformatic processing, and authentication. We describe the process all the way from processing of archaeological or historical plant material to characterizing and authenticating sequencing reads. In alternative protocols, we include modifications to this process that are tailored to strongly degraded DNA. Throughout, we stress the importance of precautionary measures to successfully analyze aDNA. Finally, we discuss the evolution of the archaeogenomics field and the development of new methods, which both shaped this protocol. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of aDNA Alternate Protocol 1: Isolation of ultra-short DNA (Dabney modification) Support Protocol 1: Preparation of PTB-based mix Support Protocol 2: Preparation of binding buffer Basic Protocol 2: Preparation of genomic libraries Alternate Protocol 2: Preparation of genomic libraries with uracil removal Basic Protocol 3: Bioinformatic processing and authentication of aDNA.
The characterization of the transcriptional similarities and differences existing between plant cells and cell types is important to better understand the biology of each cell composing the plant, to reveal new molecular mechanisms controlling gene activity, and to ultimately implement meaningful strategies to enhance plant cell biology. To gain a deeper understanding of the regulation of plant gene activity, the individual transcriptome of each plant cell needs to be established. Until recently, single cell approaches were mostly limited to bulk transcriptomic studies on selected cell types. Accessing specific cell types required the development of labor-intensive strategies. Recently, single cell sequencing strategies were successfully applied on isolated Arabidopsis thaliana root protoplasts. However, this strategy relies on the successful isolation of viable protoplasts upon the optimization of the enzymatic cocktails required to digest the cell wall and on the compatibility of fragile plant protoplasts with the use of microfluidic systems to generate single cell transcriptomic libraries. To overcome these difficulties, we present a simple and fast alternative strategy: the isolation and use of plant nuclei to access meaningful transcriptomic information from plant cells. This protocol was specifically developed to enable the use of the plant nuclei with 10× Genomics' Chromium technology partitions technology. Briefly, the plant nuclei are released from the root by chopping into a nuclei isolation buffer before purification by filtration then nuclei sorting. Upon sorting, the nuclei are resuspended in a low divalent ion buffer compatible with the Chromium technology in order to create single nuclei ribonucleic acid-sequencing libraries (sNucRNA-seq). © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis seed sterilization and planting Basic Protocol 2: Nuclei isolation from Arabidopsis roots Basic Protocol 3: Fluorescent-activated nuclei sorting (FANS) purification Support Protocol: Estimation of nuclei density using Countess II automated cell counter Alternate Protocol 1: Proper growth conditions for Medicago truncatula and Sorghum bicolor Alternate Protocol 2: Estimation of nuclei density using sNucRNA-seq technology.
Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S-acylation have been extensively studied in mammals owing to remarkable development of high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advancement of S-acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S-acylation in Arabidopsis seedlings is described. The procedures are presented in detail, including preparation of Arabidopsis seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of Arabidopsis seedling materials Basic Protocol 2: Isolation and enrichment of plasma membrane proteins Support Protocol 1: Determination of protein concentration using BCA assay Basic Protocol 3: Enrichment of S-acylated proteins by acyl-biotin exchange method Support Protocol 2: Protein precipitation by methanol/chloroform method Basic Protocol 4: Trypsin digestion and proteomic analysis Alternate Protocol: Pre-resin digestion and peptide-level enrichment.
Stress granules (SGs) are ubiquitous nonmembrane-bound assemblies of protein and mRNA formed under stress conditions associated with stalled translation. SGs are evolutionarily conserved across eukaryotes. The canonical function of SGs is to selectively protect mRNAs and proteins from unfolding and prevent degradation induced by diverse environmental stresses. Moreover, sequestration into SGs provides an elegant way to regulate protein activities. Disassembly of SGs upon stress recovery is accompanied by the reactivation of protein translation and protein activities. The regulatory importance of SGs has been corroborated by recent studies describing the multiomics analysis of the composition of SGs from yeast, animal, and plant cells. Herein, we describe an isolation protocol of SGs that allows for the identification of proteins, mRNA, and metabolites sequestered into SG cores. Furthermore, the described protocols can be used to isolate other SG-like foci. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of SG-enriched fraction from plant material Basic Protocol 2: Affinity purification to isolate SGs Basic Protocol 3: Simultaneous extraction of proteins and metabolites from affinity-purified beads Basic Protocol 4: Protein digestion on affinity-purified beads Basic Protocol 5: Data analysis.
CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants.
As the principal co-factors of many metabolic pathways, the measurement of both adenine nucleotides and nicotinamide adenine dinucleotide provides important information about cellular energy metabolism. However, given their rapid and reversible conversion as well as their relatively low concentration ranges, it is difficult to measure these compounds. Here, we describe a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides in plants. In addition, nicotinamide adenine dinucleotide is a crucially important redox-active substrate for multiple catabolic and anabolic reactions with the ratios of NAD+ /NADH and NADP+ /NADPH being suggested as indicators of the general intracellular redox potential and hence metabolic state. Here, we describe highly sensitive enzyme cycling-based colorimetric assays (with a detection limit in the pmol range) performed subsequent to a simple extraction procedure involving acid or base extraction to allow the measurement of the cellular levels of these metabolites. © 2020 The Authors. Basic Protocol 1: Preparation of plant material for the measurement Basic Protocol 2: Measurement of ATP, ADP, and AMP via HPLC Basic Protocol 3: NAD+ /NADP+ measurements Basic Protocol 4: NADH/NADPH measurements Basic Protocol 5: Data analysis and quality control approaches.
Elevation of the cytosolic free calcium ion (Ca2+ ) concentration ([Ca2+ ]cyt ) is one of the earliest responses to biotic and abiotic stress in plant cells. Among the various Ca2+ detection systems available, aequorin-based luminescence Ca2+ imaging systems provide a relatively amenable and robust method that facilitates large-scale genetic-mutant screening based on [Ca2+ ]cyt responses. Compared to that mediated by chemical elicitors, mechanical stimulation-induced elevation of [Ca2+ ]cyt is considerably more rapid, occurring within 10 s following stimulation. Therefore, its assessment using aequorin-based Ca2+ imaging systems represents a notable challenge, given that a time interval of ≥1 min is required to reduce the background light before operating the photon imaging detector. In this context, we designed a device that can rotate automatically within the confines of an enclosed dark box, and using this, we can record [Ca2+ ]cyt dynamics immediately after plants had been rotated to induce mechanical stimulation. This tool can facilitate the study of perception and early signal transduction in response to mechanical stimulation on a large scale based on [Ca2+ ]cyt responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Detection of background luminance signals in aequorin-transformed Arabidopsis seedlings using a photon imaging detector Basic Protocol 2: Construction of the rotatory device Basic Protocol 3: Calcium measurement in Arabidopsis seedlings after rotatory stimulation Basic Protocol 4: Data analysis and processing.
Plant wax lipid molecules, chiefly normal (n-) alkanes and n-alkanoic acids, are frequently used as proxies for understanding paleoenvironmental and paleoclimatic change. These are regularly analyzed from marine and lake sediments and even more frequently in archaeological contexts, enabling the reconstruction of past environments in direct association with records of past human behavior. Carbon and hydrogen isotope measurements of these compounds are used to trace plant type and water-use efficiency, relative paleotemperature, precipitation, evapotranspiration of leaf and soil moisture, and other physiological and ecological parameters. Plant wax lipids have great potential for answering questions related to human-environment interactions, being for the most part chemically inert and easily recoverable in terrestrial sediments, including those dating back millions of years. The growing use of this technique, and comparison of such data with other paleoenvironmental proxies such as pollen and phytolith analysis and soil carbonate and tooth enamel isotope records, make it essential to establish consistent, best-practice protocols for extracting n-alkanes and n-alkanoic acids from archaeological sediments to provide comparable information for interpreting past climatic, ecosystem, and hydrological changes and their interaction with human societies. © 2020 The Authors. Basic Protocol 1: Total lipid extraction Support Protocol 1: Weighing the total lipid extract Support Protocol 2: Cleaning the PSE extraction cells Alternate Protocol 1: Soxhlet total lipid extraction Alternate Protocol 2: Ultrasonic total lipid extraction Basic Protocol 2: Separation of lipids by aminopropyl column chromatography Basic Protocol 3: Separation of lipids by silver-nitrate-infused silica gel column chromatography Support Protocol 3: Preparation of silica gel infused with 10% silver nitrate Basic Protocol 4: Methylation of n-alkanoic acids Basic Protocol 5: Gas chromatography mass spectrometry (GC-MS) Basic Protocol 6: Gas chromatography isotope ratio mass spectrometry (GC-IRMS).
Root vascular pathogens are some of the world's most devastating plant pathogens. However, the methods used to determine plant susceptibility to this class of pathogen are laborious, variable, and in most cases qualitative. Here we present a rapid, simple, and robust infection assay for the characterization of Arabidopsis thaliana resistance to the fungal root pathogen Fusarium oxysporum. The method utilizes fungal root vascular penetrations and fungal-induced root growth inhibition to deliver a quantitative assessment of plant susceptibility with spatial and temporal resolution. These plant susceptibility indicators are paired with a semiautomated data analysis pipeline to deliver a reproducible assessment of plant susceptibility to root vascular pathogens such as F. oxysporum. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis thaliana plate infection assay using fluorescently labeled Fusarium oxysporum Support Protocol 1: Preparation of A. thaliana germination plates Support Protocol 2: Preparation of the F. oxysporum culture Basic Protocol 2: Data acquisition of F. oxysporum plant infection assay Support Protocol 3: Acquiring root growth inhibition data using Fiji.
Genome-editing technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Its application has been hindered in many cases, however, due to technical hurdles, such as the low rate of macromolecule delivery into plant cells and tissues or difficulties in plant transformation and regeneration. Here, a protocol for CRISPR/Cas9-based genome editing using rice zygotes is described. The genome-editing system is constructed via polyethylene glycol/calcium-mediated transfection with CRISPR/Cas9 components in rice zygotes, which are produced by in vitro fertilization of isolated rice gametes. Plasmid DNA harboring a CRISPR/Cas9 expression cassette or preassembled Cas9 protein-guide RNA ribonucleoproteins is transfected into zygotes, resulting in the regeneration of plants with a high frequency of the targeted mutation, which is either mono-allelic or bi-allelic, in the range of about 4% to 64%. Application of the present method has the potential to advance the molecular breeding of other crop species as well as rice. © 2020 Wiley Periodicals LLC.
Sorghum (Sorghum bicolor) fulfills the demand for bioenergy resources and also provides substantial diet calories to the world's population. Therefore, many biological studies use sorghum as a research model for improvement of the domesticated food and bioenergy crops. Furthermore, leveraging genome editing systems in a plethora of grass plant species has been extensively studied with no exception in sorghum. However, a protocol that details the genome editing strategies using CRISPR/Cas9 and that combines an efficient tissue culture and transformation platform in sorghum based on Agrobacterium-mediated DNA transfer has yet to be reported. This protocol outlines the steps and workflow from design of sorghum CRISPR target sites using BTx623 as a reference genome, construction of sorghum CRISPR/Cas9 plasmids, tissue culture, to Agrobacterium-mediated transformation followed by genotyping of CRISPR/Cas9 induced mutants. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 expression vector to analysis of CRISPR-edited plants Basic Protocol 2: Stable transformation of sorghum Support Protocol: Management of sorghum plants in a greenhouse.
Lipids are fascinating due to their chemical diversity, which is especially vast in the plant kingdom thanks to the high plasticity of the plant biosynthetic machinery. Lipidomic studies aim to simultaneously analyze a large number of lipid compounds of diverse classes in a given sample. The method presented here uses liquid chromatography-mass spectrometry (LC-MS)-based lipidomic profiling in a relatively fast, robust, and high-throughput manner for high-coverage quantification and annotation of lipophilic compounds. Protocols cover sample preparation, LC-MS-based measurement, and data extraction and annotation. An extensive lipid library for triacylglycerols, galactolipids, and phospholipids is provided. The extended profiling described here could be used in a range of applications and is suitable for integration with other omic datasets. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Sample preparation and metabolite extraction Basic Protocol 2: Liquid chromatography-mass spectrometry (LC-MS) analysis Basic Protocol 3: Data extraction, annotation, and quantification.
A wide variety of fresh market and processing tomatoes (Solanum lycopersicum) is grown and consumed worldwide. Post-harvest losses are a major contributing factor to losses in crop productivity and can account for up to 50% of the harvest. To select and breed elite tomato varieties, it is important to characterize fruit quality and evaluate the post-harvest properties of tomato fruits. This includes the analysis of shelf life (the period during which a fruit remains suitable for consumption without qualitative deterioration), color, and pathogen susceptibility. Tomato shelf life depends upon the rate of fruit softening which accompanies fruit ripening and exacerbates damage during transport and handling. Furthermore, the susceptibility of tomatoes to fruit pathogens is also often linked to fruit ripening, especially for necrotrophic fungi such as Botrytis cinerea, also known as gray mold. The methods described here are critical for determining fruit quality and fungal susceptibility during storage. © 2020 The Authors. Basic Protocol 1: Fruit color as a determinant of fruit quality Basic Protocol 2: Shelf life test of tomato fruits Basic Protocol 3: Botrytis cinerea pathogen test of tomato fruits Support Protocol: Preparation of Botrytis spore inoculum.
