Background

With the advancement of biotechnology, there has been a dramatic increase in the number of methods used to separate trichomes in order to study their development, biosynthesis, and metabolism within glandular trichomes. Over the years, methods of separating and collecting trichomes have made great progress in the research process [1].

Earlier methods, such as direct tweezing of trichome cells in tobacco (Nicotiana tabacum) [2] and tweezing of individual trichome cells in Arabidopsis thaliana after quick-freezing plant materials with liquid nitrogen [3] proved inefficient and inaccurate. In tomatoes (Solanum lycopersicum), Pasteur pipettes were employed to collect type VI glandular trichomes from stems and leaves [4]. Advances continued with innovations in mint (Mentha spp.), where chemical and physical methods, aided by buffer solutions, enhanced both plant material protection and glandular trichome yield [5]. These techniques have since been adapted and widely adopted across various plant species [6].

Recent years have seen the bead-beating method emerge as a predominant approach for tomato glandular trichome separation in buffered solutions, followed by filtration and density gradient centrifugation to distinguish between different glandular trichome types [7]. Similarly, this method has proven effective for enriching and purifying cannabis (Cannabis sativa) glandular trichomes [8]. Meanwhile, laser microdissection enables the direct separation of individual glandular trichomes, although its use is time-consuming, inefficient, expensive to collect large numbers of trichomes, and not suitable for large sampling requirements [9].

Previous methods, while foundational, have often suffered from limitations in sampling accuracy and efficiency, primarily focusing on glandular trichome separation while neglecting non-glandular trichomes. In addressing these limitations, this protocol focuses on utilizing North China type cucumber fruits on the day of flowering as the primary research materials. Emphasis is placed on optimizing trichome collection and separation methods to ensure adequate sampling and precise separation, encompassing both glandular and non-glandular trichomes. A substantial yield of trichomes was obtained using the bead-beating method, followed by successful separation of glandular and non-glandular trichomes using steel sieves of varying pore sizes. Non-glandular trichome contents were fully extracted via tissue grinding. Standard samples were prepared for subsequent multi-omics sequencing.

This protocol significantly enhances trichome separation efficiency, achieves precise glandular and non-glandular trichome separation, and serves as a valuable reference for trichome separation in other crops and tissues with a certain firmness. This protocol not only refines current methodologies but also underscores the critical role of precise trichome separation in advancing our understanding of plant biology and biotechnology applications.