Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

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.<.......

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.......

Materials

Name	Company	Catalog Number	Comments
Bioanalyzer RNA Pico 6000 chip	Agilent, Germany	Reorder number 5067-1513	Lab-on-a-chip system
Transsonic-310	Elma, Germany	D-78224	Ultrasonic cleaning unit
TruSeq RNA Sample Prep Kit v2	Illumina, USA	RS-122-2001	Sample preperation for RNA sequencing library
Spectrum Plant Total RNA Kit	SIGMA-ALDRICH, USA	STRN50-1KT	Plant 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, Israel	r0350n350210	Nylon screen aperture
Nylon micro-sieve with mesh size of 150 µm (size of 30 x 30 cm)	Sinun Tech, Israel	r0150n360465	Nylon screen aperture
Nylon micro-sieve with mesh size o 105 µm (size of 30 x 30 cm)	Sinun Tech, Israel	r0105n320718	Nylon screen aperture
Nylon micro-sieve with mesh size o 80 µm (size of 30 x 30 cm)	Sinun Tech, Israel	r0080n370465	Nylon screen aperture
Nylon micro-sieve with mesh size o 65 µm (size of 30 x 30 cm)	Sinun Tech, Israel	r0065n340715	Nylon screen aperture
Nylon micro-sieve with mesh size o 50 µm (size of 30 x 30 cm)	Sinun Tech, Israel	r0080n370465	Nylon 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