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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A protocol is presented for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The protocol is based on a dry, non-buffer extraction of trichomes using only liquid nitrogen, dry ice, and nylon sieves and is suitable for RNA extraction and transcriptomic analysis.

Abstract

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The biosynthetic pathways for cannabinoid and volatile terpene metabolism are localized primarily in the Cannabis trichomes, and isolated trichomes are beneficial for transcriptome analysis. The existing protocols for isolating glandular trichomes for transcriptomic characterization are inconvenient and deliver compromised trichome heads and a relatively low amount of isolated trichomes. Furthermore, they rely on expensive apparatus and isolation media containing protein inhibitors to avoid RNA degradation. The present protocol suggests combining three individual modifications to obtain a large amount of isolated glandular capitate stalked and sessile trichomes from C. sativa mature female inflorescences and fan leaves, respectively. The first modification involves substituting liquid nitrogen for the conventional isolation medium to facilitate the passage of trichomes through the micro-sieves. The second modification involves using dry ice to detach the trichomes from the plant source. The third modification involves passing the plant material consecutively through five micro-sieves of diminishing pore sizes. Microscopic imaging demonstrated the effectiveness of the isolation technique for both trichome types. In addition, the quality of RNA extracted from the isolated trichomes was appropriate for downstream transcriptomic analysis.

Introduction

Glandular trichomes are hair-like structures present in plants that contain many secondary metabolites1 and represent a valuable bank of novel biosynthetic genes and enzymes2. In Cannabis, the biosynthesis of the important secondary metabolites, cannabinoids3 and terpenes4, is localized in the trichomes. Considering the role of trichomes in determining the quality of Cannabis both for medicinal and recreational uses, the study of trichome gene expression is of interest. To characterize the expression of trichome-specific genes, the trichomes of interest mu....

Protocol

NOTE: The plant material used in this study consisted of four C. sativa ARO-Volcani strains (CS-11, CS-12, CS-13, and CS-14) that were grown in the Volcani Center, Israel, as described elsewhere11. Glandular capitate stalked trichomes were isolated from mature flowering inflorescences, and glandular capitate sessile trichomes were isolated from large fan leaves from mature non-flowering mother plants. All plant material was freshly picked and immediately stored at −80 °C.<.......

Representative Results

The main modification included in this protocol compared to conventional trichome isolation protocols is substituting the standard isolation medium with LN. Using LN as an isolation medium allows a relaxed workflow, because as long as the samples are submerged in LN, metabolic degradation is not likely to occur. Furthermore, as the protocol avoids the hazardous components (i.e., aurintricarboxylic acid and β-mercaptoethanol) used in traditional trichome isolation medium, the work is not restricted to a chemical hood.......

Discussion

Compared to the currently available trichome isolation protocols, two main modifications are described in the present protocol. These include the detachment of the trichomes from the plant material using dry ice in the initial step and substituting LN for the commonly used liquid buffer medium. The first modification for C. sativa trichome purification is based on an earlier protocol that introduced the use of crushed dry ice to detach the trichomes from geranium pedicels5. While traditio.......

Acknowledgements

The authors acknowledge financial support from CannabiVar Ltd. All plant material was generously provided by Professor Hinanit Koltai from the Volcani Center, Israel.

....

Materials

NameCompanyCatalog NumberComments
Bioanalyzer RNA Pico 6000 chipAgilent, GermanyReorder number 5067-1513Lab-on-a-chip system 
Transsonic-310Elma, GermanyD-78224Ultrasonic cleaning unit 
TruSeq RNA Sample Prep Kit v2Illumina, USARS-122-2001Sample preperation for RNA sequencing library
Spectrum Plant Total RNA Kit SIGMA-ALDRICH, USASTRN50-1KTPlant Total RNA Kit 
Nylon micro-sieve with a mesh size of 350 µm (40 x 40 cm or larger than the circumference of the flour sifter)Sinun Tech, Israelr0350n350210Nylon screen aperture
Nylon micro-sieve with mesh size of 150 µm (size of 30 x 30 cm)Sinun Tech, Israelr0150n360465Nylon screen aperture
Nylon micro-sieve with mesh size o 105 µm (size of 30 x 30 cm)Sinun Tech, Israelr0105n320718Nylon screen aperture
Nylon micro-sieve with mesh size o 80 µm (size of 30 x 30 cm)Sinun Tech, Israelr0080n370465Nylon screen aperture
Nylon micro-sieve with mesh size o 65 µm (size of 30 x 30 cm)Sinun Tech, Israelr0065n340715Nylon screen aperture
Nylon micro-sieve with mesh size o 50 µm (size of 30 x 30 cm)Sinun Tech, Israelr0080n370465Nylon screen aperture
Up to 10 g of frozen plant material (stored in -80 oC or liquid nitrogen)
Suitable gloves for handling low temperatures
Safety goggles
1 mm screen door (mosquito) mesh (strip of 30 x 100 cm)
Large strainer (colander) with holes approximately 5 mm
1 L glass beaker
1 block of dry ice (0.5-1 kg)
Hammer and hard flat object
Two 5 L plastic containers
Rubber bands
Large flour sifter or sieve strainer- preferably one with a detachable plastic ring on the circumference
Several large and small round bottom stainless steel containers. One of them should be larger than the flour sifter's circumference (approximately 40 cm in diameter), to minimize the loss of the sifted mass outside the round bottome stainless steel container
Pre-chilled (via liquid nitrogen) stainless steel spoon, spatula, and scoopula
Clean plate
Several clothespins
Pre-chilled (via liquid nitrogen) labeled 1.5 mL tubes with holes poked on the lid with a sterile needle
Two containers of liquid nitrogen
1 cm wide painting brush

References

  1. Schilmiller, A. L., Last, R. L., Pichersky, E. Harnessing plant trichome biochemistry for the production of useful compounds. Plant Journal for Cell & Molecular Biology. 54 (4), 702-709 (2008).
  2. Liu, Y., Jing, S. X., Luo, S. H., Li, S. H.

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