Glyphosate (GLY) is a widely used herbicide that can induce oxidative stress in microalgae and other non-target organisms. The quantification of GLY in surface water is a difficult task, especially in trace-level concentrations, due to its high polarity and susceptibility to biotic and abiotic degradation. Several analytical methods have been developed for GLY quantification. Most of them use high-performance liquid chromatography (HPLC) coupled with detection by mass spectrometry (MS) and include a derivatization step to decrease the polarity of the herbicide to improve detection. This protocol describes an adaptation of an existing protocol for the quantification of GLY and its metabolite aminomethylphosphonic acid (AMPA) in a water-based microalgae culture medium using ultra-high-pressure liquid chromatography (UHPLC) coupled with fluorescence detection (FLR). The principal advantage of this protocol compared with other analytical methods that employ HPLC–MS is its low cost and accessibility since it does not require an MS detector nor radioactively labeled analytical standards. Ascorbic acid (AH^-) is one of the most important hydrosoluble non-enzymatic antioxidants in eukaryotic cells and plays a key role in many metabolic pathways of critical importance in plants and algae. In this protocol, we also describe an adaptation of a previously published protocol to quantify AH^- in blood samples to be used in microalgal cells exposed to GLY and GLY-based herbicides. The sample preparation procedure for this last protocol is fast, easy, and does not require expensive equipment. It uses an HPLC system coupled with an electrochemical detector (EC) for AH^- quantification but may be adapted to be used with a UV-Vis detector.

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.

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.

The Fusarium genus includes various fungi of great significance in agriculture. Fusarium solani f. sp. eumartii (F. eumartii), traditionally known as a potato pathogen, has also been identified as a cause of disease in tomatoes. This protocol provides a detailed, efficient, and robust flood-inoculation method for assessing F. eumartii infection of young tomato seedlings grown on MS medium plates. It includes the evaluation of the lesion area and the quantification of the remaining fungal inoculum in tomato seedlings. In summary, the straightforwardness and efficiency of this bioassay make it a powerful quantitative tool for selecting fungicidal compounds or defense response inducers in tomato plants, offering a promising approach with significant potential for preventing fungal diseases in crops.

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.

Diatoms serve as a source for a variety of compounds with particularbiotechnological interest. Therefore, redirecting the flow to a specific pathwayrequires the elucidation of the gene’s specific function. The mostcommonly used method in diatoms is biolistic transformation, which is a veryexpensive and time-consuming method. The use of episomes that are maintained asclosed circles at a copy number equivalent to native chromosomes has become auseful genetic system for protein expression that avoids multiple insertions,position-specific effects on expression, and potential knockout of non-targetedgenes. These episomes can be introduced from bacteria into diatoms viaconjugation. Here, we describe a detailed protocol for gene expression thatincludes 1) the gateway cloning strategy and 2) the conjugation protocol for themobilization of plasmids from bacteria to diatoms.