Plant Science

CRISPR/Cas9 genome editing technology has revolutionized plant breeding by offering precise and rapid modifications. Traditional breeding methods are often slow and imprecise, whereas CRISPR/Cas9 allows for targeted genetic improvements. Previously, direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts has been demonstrated, but successful regeneration of edited protoplasts into whole plants has not been achieved. Here, we describe an efficient protocol for obtaining transgene/DNA-free edited grapevine plants by transfecting protoplasts isolated from embryogenic callus and subsequently regenerating them. The regenerated edited plants were comparable in morphology and growth habit to wild-type controls. This protocol provides a highly efficient method for DNA-free genome editing in grapevine, addressing regulatory concerns and potentially facilitating the genetic improvement of grapevine and other woody crop plants.

In applications such as marker-assisted breeding and positional cloning, tissue sampling and plant tracking are vital steps in the genotyping pipeline. They enable the identification of desirable seedlings, saving time and reducing the cost, space, and handling required for growing adult plants, especially for greenhouses and winter nurseries. Small-scale marker-assisted selection laboratories rely heavily on leaf-based genotyping, which involves over-planting large, segregating populations followed by leaf sampling, genotyping, and backtracking to identify desired individuals, which is costly and laborious. Thus, there is a need to adopt seed-based genotyping to reduce costs and save time. Therefore, we developed a safe and cheap seed-chipping protocol using clipping pliers to chip seeds to genotype before planting. To identify a cost-effective and high-throughput DNA extraction method, we tested four extraction methods and assessed the quality of the seed DNA using PCR. For three of the methods, seed-based DNA was of comparable quality to DNA extracted from leaf punches. We also compared seed- and leaf-derived DNA from the same individuals in a segregating population to test for genotyping miscalls that could arise due to the presence of maternally derived pericarp in the seed samples. Out of 43 potential instances, we found zero miscalled samples and, therefore, no evidence supporting consequential pericarp inclusion. Germination rates of chipped and unchipped seeds were the same for the inbreds tested, B73 and Mo17. However, chipped seeds grew slower until ~14 days after sowing. Overall, seed sampling using clipping pliers provides a simple, reliable, and high-throughput method to identify specific genotypes before planting.

Improving desirable traits of popular rice varieties is of particular importance for small-scale food producers. Breeding is considered the most ecological and economic approach to improve yield, especially in the context of pest and pathogen-resistant varieties development. Being able to cross rice lines is also a critical step when using current transgene-based genome editing technologies, e.g., to remove transgenes. Moreover, rice breeders have developed accelerated breeding methods, including marker-assisted backcross breeding (MABB) to develop novel rice varieties with in-built resistance to biotic and abiotic stressors, grain, and nutritional quality. MABB is a highly efficient and cost-effective approach in accelerating the improvement of recipient variety by introgressing desirable traits, especially from landrace cultivars and wild rice accessions. Here, we provide a detailed protocol including video instructions for rice crossing and MABB to introgress target trait(s) of interest into the elite rice line. Further, we also highlight tips and tricks to be considered for a successful crossing and MABB.

In recent years, the increase in genome sequencing across diverse plant species has provided a significant advantage for phylogenomics studies, allowing the analysis of one of the most diverse gene families in plants: nucleotide-binding leucine-rich repeat receptors (NLRs). However, due to the sequence diversity of the NLR gene family, identifying key molecular features and functionally conserved sequence patterns is challenging through multiple sequence alignment. Here, we present a step-by-step protocol for a computational pipeline designed to identify evolutionarily conserved motifs in plant NLR proteins. In this protocol, we use a large-scale NLR dataset, including 1,862 NLR genes annotated from monocot and dicot species, to predict conserved sequence motifs, such as the MADA and EDVID motifs, within the coiled-coil (CC)-NLR subfamily. Our pipeline can be applied to identify molecular signatures that have remained conserved in the gene family over evolutionary time across plant species.

CRISPR-Cas9 technology has become an essential tool for plant genome editing. Recent advancements have significantly improved the ability to target multiple genes simultaneously within the same genetic background through various strategies. Additionally, there has been significant progress in developing methods for inducible or tissue-specific editing. These advancements offer numerous possibilities for tailored genome modifications. Building upon existing research, we have developed an optimized and modular strategy allowing the targeting of several genes simultaneously in combination with the synchronized expression of the Cas9 endonuclease in the egg cell. This system allows significant editing efficiency while avoiding mosaicism. In addition, the versatile system we propose allows adaptation to inducible and/or tissue-specific edition according to the promoter chosen to drive the expression of the Cas9 gene. Here, we describe a step-by-step protocol for generating the binary vector necessary for establishing Arabidopsis edited lines using a versatile cloning strategy that combines Gateway^® and Golden Gate technologies. We describe a versatile system that allows the cloning of as many guides as needed to target DNA, which can be multiplexed into a polycistronic gene and combined in the same construct with sequences for the expression of the Cas9 endonuclease. The expression of Cas9 is controlled by selecting from among a collection of promoters, including constitutive, inducible, ubiquitous, or tissue-specific promoters. Only one vector containing the polycistronic gene (tRNA-sgRNA) needs to be constructed. For that, sgRNA (composed of protospacers chosen to target the gene of interest and sgRNA scaffold) is cloned in tandem with the pre-tRNA sequence. Then, a single recombination reaction is required to assemble the promoter, the zCas9 coding sequence, and the tRNA-gRNA polycistronic gene. Each element is cloned in an entry vector and finally assembled according to the Multisite Gateway^® Technology. Here, we detail the process to express zCas9 under the control of egg cell promoter fused to enhancer sequence (EC1.2en-EC1.1p) and to simultaneously target two multiple C2 domains and transmembrane region protein genes (MCTP3 and MCTP4, respectively at3g57880 and at1g51570), using one or two sgRNA per gene.

Polysome profiling by sucrose density gradient centrifugation is commonly used to study the overall degree of translation (messenger RNA to protein synthesis). Traditionally, the method begins with synthesis of a 5–10 mL sucrose gradient onto which 0.5–1 mL of cell extract is layered and centrifuged at high speed for 3–4 h in a floor-model ultracentrifuge. After centrifugation, the gradient solution is passed through an absorbance recorder to generate a polysome profile. Ten to twelve fractions (0.8–1 mL each) are collected for isolating different RNA and protein populations. The overall method is tedious and lengthy (6–9 h), requires access to a suitable ultracentrifuge rotor and centrifuge, and requires a substantial amount of tissue material, which can be a limiting factor. Moreover, there is often a dilemma over the quality of RNA and protein populations in the individual fractions due to the extended experiment times. To overcome these challenges, here we describe a miniature sucrose gradient for polysome profiling using Arabidopsis thaliana seedlings that takes ~1 h centrifugation time in a tabletop ultracentrifuge, reduced gradient synthesis time, and also less tissue material. The protocol described here can be easily adapted to a wide variety of organisms and polysome profiling of organelles, such as chloroplasts and mitochondria.

Key Features

• Mini sucrose gradient for polysome profiling that requires less than half the processing time vs. traditional methods.

• Reduced starting tissue material and sample volume for sucrose gradients.

• Feasibility of RNA and protein isolation from polysome fractions.

• Protocol can be easily modified to a wide variety of organisms (and even polysome profiling of organelles, such as chloroplast and mitochondria).

Graphical Overview

Figure 1. Graphical overview of polysome profiling using mini sucrose gradient. A. One milliliter each of 15% (w/v) and 50% (w/v) sucrose gradient solution is added to the individual chambers of the gradient maker. While mixing with a small magnetic stirrer in the 50% solution chamber, base station knob is turned to open position, allowing sucrose gradient solution to slowly flow through the outlet into a 2.2 mL gradient tube. After centrifugation at 50,000 rpm (213,626.2 × g) in a swinging bucket rotor for 70 min at 4 °C, the gradient tube is stored at 4 °C for the next steps. B. Cell extract from 12-day-old vertically grown Arabidopsis thaliana seedlings is centrifuged twice and 100 µL of supernatant is gently layered on the pre-made sucrose gradient from step A. After centrifugation as described in step A, polysome profile is obtained by feeding the gradient solution through an absorbance recorder (A254 nm). Eight (200 µL) fractions are collected for RNA and protein isolation.

CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing. In this protocol, we describe the detailed step-by-step instructions for using this system.

CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.

Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, the inheritance of specific methylation patterns can be studied. Here, we present a protocol for analyzing DNA methylation patterns through MSAP markers in potato interspecific hybrids and their parental genotypes.

Homologous genes, including paralogs and orthologs, are genes that share sequence homologies within or between different species. Homologous genes originate from a common origin through speciation, genetic duplication or horizontal gene transfer. Estimation of the sequence divergence of homologous genes help us to understand divergence time, which makes it possible to understand the evolutionary patterns of speciation, gene duplication and gene transfer events. This protocol will provide a detailed bioinformatics pipeline on how to identify the homologous genes, compare their sequence divergence and phylogenetic relationships, focusing on homologous genes that show syntenic relationships using soybean (Glycine max) and common bean (Phaseolus vulgaris) as example species.