植物科学

Gene stacking, the process of introducing multiple genes into a single plant to enhance desired traits, is essential for plant genetic improvement through both conventional breeding and genetic transformation. In general, transformation-based gene stacking can be achieved through either co-transformation to simultaneously introduce multiple genes or sequential multi-round transformation. While co-transformation is generally faster and more efficient than sequential multi-round transformation, it often requires two selectable marker genes, which confer resistance to antibiotics, for selecting transgenic events. However, in most cases, there is only one best selectable marker gene for a specific plant species or genotype. Also, it is harder to optimize the concentrations of two antibiotics for co-transformation than using one antibiotic for selecting transgenic events. To overcome this challenge, we recently developed an innovative split selectable marker system for plant co-transformation, allowing the use of one selectable marker gene to select transgenic events. This method involves constructing two binary vectors, each carrying a subset of genes of interest and a partial fragment of the selectable marker gene, which is connected to a partial intein fragment. Following Agrobacterium-mediated co-transformation, plants harboring both binary vectors are selected using a single antibiotic, such as kanamycin. This split-marker system can be used to co-transform multiple genes into both herbaceous and woody plants, accelerating genetic improvement of polygenic traits or integrative improvement of multiple traits to simultaneously increase crop yield and quality.

Agrobacterium-mediated gene transformation method is a vital molecular biology technique employed to develop transgenic plants. Plants are genetically engineered to develop disease-free varieties, knock out unsettling traits for crop improvement, or incorporate an antigenic protein to make the plant a green factory for edible vaccines. The method’s robustness was validated through successful transformations, demonstrating its effectiveness as a standard approach for researchers working in plant biotechnology. It enables the introduction of foreign DNA into plant genomes. Conventionally, plant genetic transformation has relied on time-consuming, costly, and technically demanding procedures, such as electroporation and chimeric viruses or biolistic methods, which usually yield variable transformation efficiencies. This study presents a simple and fail-safe protocol that involves a modified freeze-thaw and heat-shock concoction method. This approach involves a streamlined plasmid miniprep procedure to isolate high-quality plasmid DNA from Escherichia coli K12 strain, followed by a target-specific transfer into A. tumefaciens EHA105 strain. The optimized method minimizes DNA degradation and maximizes uptake by Agrobacterium cells, making it a reproducible and accessible protocol for various genetic engineering applications. The transformation efficiency is consistently high, enhancing plasmid uptake while maintaining cell viability, requiring minimal specialized equipment and reagents. The proposed protocol offers significant advantages, including simplicity, reliability, and cost-effectiveness, positioning it as a valuable alternative to traditional techniques in the field of plant biotechnology.

Gene expression analysis is a fundamental technique to elucidate the regulatory mechanisms of genes of interest or to reveal the patterns of plant response to environmental stimuli. Traditionally, gene expression analyses have required RNA extraction, followed by cDNA synthesis and qPCR analyses. However, this conventional method is costly and time-consuming, limiting the amount of data collected. The protocol outlined in this study, which utilizes a chemiluminescence system, offers a cost-effective and rapid method for assessing the expression of Arabidopsis (Arabidopsis thaliana) genes, exemplified by analyzing the nitrate-inducible expression of a major nitrate transporter gene, nitrate transporter 2.1 (NRT2.1). A reporter construct, containing the NRT2.1 promoter fused to the firefly luciferase gene, was introduced into wild-type and mutant Arabidopsis plants. Seeds obtained from the transgenic lines were grown for 3 days in 96-well microplates containing a nitrate-free nutrient solution. After 3 days, the nutrient solution was replaced with a fresh batch, which was supplemented with luciferin potassium. One hour later, nitrate was added at various concentrations, and the temporal expression pattern of NRT2.1 was analyzed by monitoring the chemiluminescence signals. This method allowed for the cost-effective, quantitative, and high-throughput analysis of NRT2.1 expression over time under the effects of various nutrient conditions and genetic backgrounds.

Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.

Cloning systems like Gateway and Golden Gate/Braid are known because of their efficiency and accuracy. While the main drawback of Gateway is the expensive cost of the enzymes used in its two-step (LR and BP) reaction, Golden Gate requires non-reusable components due to their specific restriction sites. We present the Brick into the Gateway (BiG) protocol as a new cloning strategy, faster and more economic method that combines (i) reusable modules or bricks assembled by the GoldenBraid approach, and (ii) Gateway LR reactions [recombination of attachment sites: attL (L from left) and attR (R from right)] avoiding the BP reaction [recombination of attachment sites: attP (P from phage) and attB (B from bacteria)] usually necessary in the Gateway cloning. The starting point is to perform a PCR reaction to add type IIS restriction sites into DNA fragments generating specific fusion sites. Then, this PCR product is used to design GoldenBraid bricks, including the attL Gateway recombination sites. Using the Golden Gate method, these bricks are assembled to produce an attL1–gene of interest–attL2 fragment, which is integrated into a compatible vector producing a Gateway entry vector. Finally, the fragment containing the target gene is recombined by LR reaction into the Gateway destination vector.

Graphical abstract

Plant genomes are pronouncedly enriched in repeat elements such as transposons. These repeats are epigenetically regulated by DNA methylation. Whole genome high-depth sequencing after bisulfite treatment remains an expensive and laborious method to reliably profile the DNA methylome, especially when considering large genomes such as in crops. Here, we present a simple reproducible Southern hybridisation–based assay to obtain incontrovertible methylation patterns from targeted regions in the rice genome. By employing minor but key modifications, we reliably detected transposon copy number variations over multiple generations. This method can be regarded as a gold standard for validation of epigenetic variations at target loci, and the consequent proliferation of transposons, or segregation in several plant replicates and genotypes.

Graphical abstract:

Gene expression depends on the binding of transcription factors with DNA regulatory sequences. The level of accessibility for these sequences varies between cells and cell types. Until recently, using the Tn5 assay for transposase-accessible chromatin for sequencing (ATAC-seq) technology allowed assessing the profiles of chromatin from an entire organ or, when coupled with the isolation of nuclei tagged in specific cell types (INTACT) method, from a cell-type. Recently, ATAC-seq experiments were conducted at the level of individual plant nuclei. Applying single nuclei ATAC-seq (sNucATAC-seq) technology to thousands of individual cells revealed more finely tuned profiles of chromatin accessibility. In this manuscript, we describe a method to isolate nuclei fom plant roots and green tissues, permeabilize the nuclear membrane using detergent to allow the penetration of the Tn5 transposase, and re-suspend them in a nuclei resuspension buffer compatible with the construction of sNucATAC-seq libraries using the 10× Genomic’s Chromium technology. This protocol was successfully applied on Arabidopsis thaliana and Glycine max root nuclei.

Transgenic plants are produced both to investigate gene function and to confer desirable traits into crops. Transgene copy number is known to influence expression levels, and consequently, phenotypes. Similarly, knowledge of transgene zygosity is desirable for making quantitative assessments of phenotype and tracking the inheritance of transgenes in progeny generations. Since the first transgenic plants were produced, several methods for determining copy number have been applied, including Southern blotting, quantitative real-time PCR, and more recently, sequencing methods; however, each method has specific disadvantages, compromising throughput, accuracy, or expense. Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of occurrence of specific sequences to be accurately estimated. Confidence increases with the number of partitions; therefore, the availability of emulsion technologies that enable reactions to be divided into tens of thousands of nanodroplets allows accurate determination of copy number in what has become known as digital droplet PCR (ddPCR). ddPCR offers similar benefits of low costs and scalability as other PCR techniques but with superior accuracy and reliability.

Graphic abstract:

Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of transgene copy number to be accurately assessed.

This protocol describes the generation of protoplasts from protonemal tissue of the moss Physcomitrium patens (syn. Physcomitrella patens), using Cellulase ONOZUKA R10 and Macerozyme R10, followed by polyethylene glycol (PEG) mediated transformation. The protonemal tissue grown in liquid suspension was harvested and treated with enzyme cocktails mix of 1.5% Cellulase ONOZUKA R10 and 0.5% Macerozyme R10 to generate 1,8 million protoplasts within 3 h.

Gene knock-down in plants is a useful approach to study genotype-phenotype relationships, render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small interfering RNA (siRNA)-mediated gene silencing is one of the most common ways to achieve gene knock-down in plants. Traditionally, siRNA is delivered into intact plant cells by coding the siRNA sequences into DNA vectors, which are then delivered through viral and/or bacterial methods. In this protocol, we provide an alternative direct delivery method of siRNA molecules into intact plant cells for efficient transient gene knock-down in model tobacco plant, Nicotiana benthamiana, leaves. Our approach uses one dimensional carbon-based nanomaterials, single-walled carbon nanotubes (SWNTs), to deliver siRNA, and does not rely on viral/bacterial delivery. The distinct advantages of our method are i) there is no need for DNA coding of siRNA sequences, ii) this abiotic method could work in a broader range of plant species than biotic methods, and iii) there are fewer regulatory complications when using abiotic delivery methods, whereby gene silencing is transient without permanent modification of the plant genome.

Graphic abstract