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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 must first be isolated. Trichome isolation protocols were first described as early as 1992⁵, and their latest developments have been recently reviewed². In general, protocols for extracting glandular trichomes for transcriptomic characterization can be divided into two distinct sequential steps. The first step involves a thorough physical separation of the trichomes from the plant tissue. This step can be performed by using dry ice⁵, glass beads with a commercial apparatus^6,7, grinding the plant material against a mesh sieve⁸, or vortexing the plant tissue in an isolation buffer⁹. The second step involves a more refined separation of the trichomes of interest from the microscopic plant residue and/or other trichome types. This step can be executed using density gradient centrifugation^8,10 or sieves of various sizes^7,9. Due to the extreme sensitivity of RNA in processed tissues to degrading agents, these two sequential steps are usually conducted in the ice-cold isolation medium, often in the presence of protein inhibitors⁴.

Conventional trichome isolation protocols require, in addition to the ice-cold temperatures, large amounts of isolation medium to ensure an efficient extraction procedure. The combination of these components results in an arduous, time-consuming isolation process that hinders high throughput. Presenting a straightforward, user-friendly alternative trichome isolation protocol is, therefore, likely to be beneficial for various aspects associated with trichome characterization. The present paper aims to offer an alternative protocol for isolating stalked and sessile glandular capitate trichomes from Cannabis sativa by combining and integrating several elements from the conventional protocols. These elements include dry ice⁵, the passing of the trichomes through several micro-sieves with decreasing pore sizes^7,9, and substituting liquid nitrogen (LN) for the isolation medium⁸.

The novelty of the present trichome isolation protocol, as compared to conventional protocols, presents in a number of ways. This protocol is convenient, as it does not require hazardous components. The procedure can be conducted in the lab with minimal precautions and facilitates high throughput. Substituting LN for the standard liquid isolation medium ensures the integrity of the trichomes throughout the isolation process, enabling subsequent transcriptomic analysis. Upon sublimation of the LN and dry ice, the isolated trichomes are left free of harmful residuals. Further, the propensity of LN to sublimate at room temperature allows its generous use throughout the protocol. In contrast, using large volumes of conventional isolation medium generates practical difficulties in its handling. Finally, the protocol decreases the separation of the disc cell from the remaining fragile head structure of the glandular trichome, enabling the retention of the headspace content.

This protocol is presented in a detailed step-by-step fashion designed to assist the technical practice of isolating C. sativa glandular capitate trichomes. The protocol provides a manageable workflow that results in isolated trichomes with a high concentration and purity that are appropriate for downstream molecular analysis.

Protokół

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.

CAUTION: Dry ice and LN are used throughout the protocol. These substances are extremely hazardous. The isolated trichomes can contain dry ice particles that can create dangerous gas pressure when inserted into sealed tubes; therefore, all caps should be needle-punctured. The use of protective goggles, appropriate lab wear, and gloves for handling at extremely low temperatures is highly advised.

1. Setup for the initial separation of trichomes from the plant material

1. Crush a dry ice block into small fine flakes using a hammer and a hard flat object in a 5 L plastic container.
2. Sieve the fine dry ice flakes (smaller than 5 mm) from the non-crushed dry ice with a large strainer (via pores smaller than 5 mm) into another 5 L plastic container. Pour approximately 200 cm³ (200 mL mark) of the dry ice flakes into an upright 1 L glass beaker.
3. Add up to 10 g of frozen C. sativa inflorescences (from now on referred to as plant material) onto the first layer of crushed dry ice, and cover with an additional layer of 200 cm³ of finely crushed dry ice.
4. Cover the opening of the 1 L glass beaker with two to three layers of 1 mm screen door mosquito net, and secure it to the outer sides of the glass cup with rubber bands (Figure 1).
5. Pour LN into a large round-bottom stainless steel container (see Table of Materials), where the isolated trichomes will be collected.
6. Insert a 350 µm mesh in a flour sifter/sieve strainer to cover the flour sifter mesh from below (Figure 2). If a flour sifter with a detachable plastic ring is available, insert the 350 µm mesh so that it is secured under the flour sifter's mesh. If not, secure the 350 µm mesh to the circumference of the flour sifter with rubber bands.
7. Place the flour sifter above the large round-bottom stainless steel container filled with LN (Figure 3). Ensure that the width of the container exceeds that of the flour sifter to minimize the loss of the sifted mass outside the round-bottom stainless steel container.

2. Separation of trichomes from the plant material

1. Shake the 1 L glass with its opening pointing downward toward the flour sifter as if using a large salt shaker (Figure 4).
2. Set aside the 1 L glass beaker every 2-3 min to allow the sifting of the crushed dry ice and plant material accumulated on the flour sieve.
3. Sift the flour sieve horizontally to facilitate the passage of the plant material into the LN in the round-bottom stainless steel container below.
4. Add LN to the stainless steel container and crushed dry ice to the 1 L glass beaker when their levels run low. Replenish the used plant material in the glass beaker with an additional 10 g of fresh plant material when the plant material on the flour sieve is depleted. The replenishment usually does not have to be repeated more than twice.
5. Repeat steps 2.1-2.4 until a sufficient amount of enriched trichomes has been collected in the round-bottom stainless steel container. Generally, 20 g of fresh plant material is sufficient for RNA extraction and transcriptome analysis.
6. Verify that there is a white powder-like substance (consisting of plant debris, glandular trichomes, and crushed dry ice) at the bottom of the stainless steel container submerged in the LN. A microscopic observation will help to determine if a sufficient amount of trichomes has been collected; however, this step might obstruct the protocol's flow. Usually, 10-20 g of initial plant material suffices for most isolations; however, there may be differences in the trichome density of the plants.
   NOTE: From this point, the experiment can be paused and restarted later. If paused for a short time, adequate amounts of LN should be added so that the trichomes remain submerged. Alternatively, the trichomes can be collected and stored at −80 °C for up to 3 months for later use, as described in step 5.

3. Separation of glandular capitate trichomes (stalked and sessile) from other trichome types, such as bulbous and cystolithic trichomes, and debris

1. Add a small amount of LN to a clean small round-bottom stainless steel container.
2. Fold a 40 cm x 40 cm micro-sieve with a 150 µm mesh size twice to obtain a 20 cm x 20 cm fold, and open it. Ensure it resembles a cone-like shape (Figure 5).
3. Secure the mesh cone to the edge of the round-bottom stainless steel container with one or two clothespins so that the opened part of the cone is upright and its pointed part is partly submerged in the LN.
4. Gently pour the LN and the white powder-like substance from step 2.6 into the micro-sieve cone.
5. Gently apply a wide brush to gather and transfer any remaining plant material from the first container into the 150 µm mesh cone.
6. Add more LN to the first container, and repeat the brushing motion until all the plant material is transferred to the micro-sieve cone.
7. Carefully detach the clothespin from the lid of the container, and open the micro-sieve cone so that the trichomes are located in the middle of the opened micro-sieve. Hold all four corners of the micro-sieve together so that the middle part containing the trichomes remains submerged in the LN (Figure 6).
8. While holding all four corners of the micro-sieve, gently dip and shake the micro-sieve in the LN in the steel container as if infusing a tea bag. Continue this dipping/shaking (vertical and horizontal) motion for 1 min. There is a tradeoff between the isolation quality and quantity. Longer dipping motions may result in larger amounts of trichomes, but their isolation grade may be poorer. Overall, 1 min is recommended as adequate for both quality and quantity.
   NOTE: This sieving motion is the most critical part of the protocol and should be executed accordingly.
9. Optional: Scoop the non-sifted plant debris and larger trichomes (still submerged in the LN) that are wrapped in the center of the micro-sieve into a 13 mL or 50 mL test tube, and store at −80 °C for future use.
   ​CAUTION: To avoid the buildup of pressured gas (from the dry ice and LN residues), puncture a hole in the cap of the test tube with a sterilized needle. The cap can be replaced once the pressure has reduced, which is usually after 24 h.

4. Passing trichomes through micro-sieves of decreasing pore size

1. Transfer the sifted trichomes submerged in the LN at the bottom of the small round-bottom stainless steel container sequentially through micro-sieves with decreasing pore sizes (105 µm, 80 µm, 65 µm, and 50 µm), in the same manner as presented in step 3.

5. Collection of the desired trichomes

1. Remove the desired trichomes submerged in the LN and wrapped in the 50 µm micro-sieve using a pre-chilled (in LN) spoon. Place them on a clean plate.
   NOTE: In this final isolation stage, the desired trichomes are collected from the sieve.
2. Quickly transfer the powder-like trichomes into a labeled pre-chilled 1.5 mL tube via a pre-chilled spatula or a scoopula inserted into the tube (Figure 7). Store the tubes immediately at −80 °C for further studies.
   ​NOTE: A schematic workflow chart depicting the whole isolation protocol is given in Figure 8.

6. Observation and analysis of the purified trichomes

1. Place a small amount (10 mg) of isolated trichomes on a microscope slide using a pre-cooled (via LN) spatula. Add a drop of water, and directly observe on a light microscope without staining. Evaluate the overall isolation and contamination degree using low (10x) and high (40x) magnifications. Enrichment is visually estimated by the relative amount of trichomes with respect to non-trichome tissue, as shown in Figure 9.
2. Perform RNA extraction and quality analysis from 50 mg of isolated C. sativa glandular capitate stalked and sessile trichomes.
   NOTE: A commercial extraction kit (see Table of Materials) employing a poly-A-based strategy of mRNA purification was used for the RNA extraction.
   1. Resuspend 50 mg of the isolated trichomes in the lysis solution, vortex for 30 s, sonicate in an ice-cold ultrasonic cleaning unit at 35 kHz for 5 min, and extract the mRNA from the samples according to the manufacturer's protocol (see Table of Materials).
   2. To estimate the integrity of the RNA extracted from the trichomes, determine the RNA concentration, the total amount, and the RNA integrity number (RIN) values using a lab-on-a-chip system (see Table of Materials).
   3. Perform RNAseq analysis of the trichome fractions (optional). RNAseq analysis and bioinformatics analyses of sequenced reads can be performed by standard outsourcing services

Wyniki

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...

Dyskusje

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...

Materiały

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