植物科学

Extracellular vesicles (EVs) are membrane-bound, non-replicating particles released by virtually all types of cells. EVs concentrate and deliver a plethora of biomolecules driving very important biological functions, including intercellular communication not only between cells of the same organism but also across different kingdoms. Plant extracellular vesicles (PEVs) are a promising alternative to mammalian EVs in biomedical applications. Here, we present an optimized and reproducible protocol for isolating PEVs from the hairy root (HR) cultures of medicinal plants Salvia dominica and S. sclarea. Our methodological approach introduces a significant advancement in the standardization of HR-EVs purification processes from plant biotechnological platforms, paving the way for their broader application across various sectors, including agriculture, pharmaceuticals, and nutraceuticals.

Plant proteases participate in a wide variety of biological processes, including development, growth, and defense. To date, numerous proteases have been functionally identified through genetic studies. However, redundancy among certain proteases can obscure their roles, as single-gene loss-of-function mutants often exhibit no discernible phenotype, limiting identification through genetic approaches. Here, we describe an efficient system for the identification of target proteases that cleave specific substrates in the Arabidopsis apoplastic fluid. The method involves using Arabidopsis-submerged culture medium, which contains apoplastic proteases, followed by native two-dimensional electrophoresis. Gel fractionation and an in-gel peptide cleavage assay with a fluorescence-quenching peptide substrate are then used to detect specific proteolytic activity. The active fraction is then subjected to mass spectrometry–based proteomics to identify the protease of interest. This method allows for the efficient and comprehensive identification of proteases with specific substrate cleavage activities in the apoplast.

Seed germination is a critical and challenging process in the propagation of Oryza coarctata, a wild halophytic rice species. This protocol outlines the seed germination procedure for O. coarctata. All steps required for optimal germination and seedling establishment of O. coarctata in both sterile and soil-based systems are described in detail. Additionally, the protocol includes an analysis of the primary hormones, abscisic acid (ABA) and gibberellin (GA), involved in regulating seed dormancy and germination.

Cannabis (Cannabis sativa L.) derivatives are of great importance in the medical, cosmetic, and pharmaceutical industries. This relevance is mainly due to the active principles (cannabinoids) found mainly in the trichomes of the female inflorescences. One of the most commonly used methods to propagate cannabis is by vegetative stem cuttings. This low-cost technique produces genetically uniform plants, ensuring consistent growth rates and cannabinoid production. The extraction of cannabinoids and other active compounds from the resin of the flowers is the main limitation of cannabis processing. Here, we describe a step-by-step protocol for propagating female cannabis plants from vegetative stem cuttings, inducing flower development, and obtaining high-quality cannabinoid-enriched resin.

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.

To prepare Hevea brasiliensis plantations, selected planting material is propagated by grafting using illegitimate seedlings as rootstocks, whose paternal genotype is unknown. Recent advances in rubber tree in vitro cloning propagation open the possibility of using these techniques to supply new planting material. Micrografting is a promising technique to speed up the preparation of plant material for rootstock–scion interaction studies. This article describes the implementation of an efficient micrografting technique from Hevea in vitro plants from clone PB 260. The procedure combines several conditions to preserve the root system and the grafted scion and to prevent any breakage of rootstock buds. This technique paves the way for clonal propagation and holds potential for further development on other rubber clones for further studies on the interaction between rootstock and scion.

In nature, filamentous fungi interact with plants. These fungi are characterized by rapid growth in numerous substrates and under minimal nutrient requirements. Investigating the interaction of these fungi with their plant hosts under controlled conditions is of importance for many researchers aiming to proceed with molecular or microscopical investigations of their favorite plant–fungus interaction system. The speed of growth of these fungi complicates transferring plant–fungal interaction systems in laboratory conditions. The issue is more complicated when monoxenic conditions are desired, to ensure that only two members (a fungus and a plant) are present in the system under study. Here, two simple closed systems for investigating plant–filamentous fungi associations under laboratory, monoxenic conditions are described, along with their limitations. The plant and fungal growth conditions, methods for sampling, staining, sectioning, and subsequent microscopical imaging of colonized plant tissues with affordable, common laboratory tools are described.

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.

Plant embryos are contained within seeds. Isolating them is crucial when endosperm and seed coat tissues interfere with the study of mutant genetic functions due to differing genotypes between maternal and embryonic tissues. RNA extraction from plant embryonic tissue presents particular challenges due to the high activity of RNases, the composition of the seed, and the risk of RNA degradation. The developmental stage of the embryo is a key aspect of successful isolation and RNA extraction due to the size and amount of tissue. Proper handling during RNA extraction is critical to maintain RNA integrity and prevent degradation. While commercial kits offer various methods for RNA extraction from embryos, homemade protocols provide valuable advantages, including cost-effectiveness and accessibility for labs with limited funding. Here, we present a simple and efficient protocol for extracting RNA from isolated Arabidopsis thaliana embryos at the torpedo/cotyledon stage using a homemade extraction buffer previously reported for styles of Nicotiana alata.

Understanding how multicellular organisms are shaped requires high-resolution, quantitative data to unravel how biological structures grow and develop over time. In recent years, confocal live imaging has become an essential tool providing insights into developmental dynamics at cellular resolution in plant organs such as leaves or meristems. In the context of flowers, growth tracking has primarily been limited to sepals, the outermost floral organs, or the post-fertilization gynoecium, which are easily accessible for microscopy. Here, we describe a detailed pipeline for the preparation, dissection, and confocal imaging of the development of internal reproductive floral organs of Arabidopsis thaliana including both the stamen and gynoecium. We also discuss how to acquire high-quality images suitable for efficient 2D and 3D segmentation that allow the quantification of cellular dynamics underlying their development.