Name: non-aqueous isolation and enrichment of glandular capitate stalked and sessile trichomes from cannabis sativa
Uploaded: 2023-05-12T12:30:00
Description: Watch this Scientific Journal Video about Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa at JoVE.com

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

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 &#8722;80&#160;&#176;C.<...

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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...

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 apparatus6,7, 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 centrifugation8,10 or sieves of various sizes7,9. 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 sizes7,9, and substituting liquid nitrogen (LN)&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bioanalyzer RNA Pico 6000 chip</td>
			<td>Agilent, Germany</td>
			<td>Reorder number 5067-1513</td>
			<td>Lab-on-a-chip system&#160;</td>
		</tr>
		<tr>
			<td>Transsonic-310</td>
			<td>Elma, Germany</td>
			<td>D-78224</td>
			<td>Ultrasonic cleaning unit&#160;</td>
		</tr>
		<tr>
			<td>TruSeq RNA Sample Prep Kit v2</td>
			<td>Illumina, USA</td>
			<td>RS-122-2001</td>
			<td>Sample preperation for RNA sequencing library</td>
		</tr>
		<tr>
			<td>Spectrum Plant Total RNA Kit&#160;</td>
			<td>SIGMA-ALDRICH, USA</td>
			<td>STRN50-1KT</td>
			<td>Plant Total RNA Kit&#160;</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with a mesh size of 350 &#181;m (40 x 40 cm or larger than the circumference of the flour sifter)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0350n350210</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size of 150 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0150n360465</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size o 105 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0105n320718</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size o 80 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0080n370465</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size o 65 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0065n340715</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size o 50 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0080n370465</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Up to 10 g of frozen plant material (stored in -80 oC or liquid nitrogen)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suitable gloves for handling low temperatures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Safety goggles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm screen door (mosquito) mesh (strip of 30 x 100 cm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large strainer (colander) with holes approximately 5 mm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 L glass beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 block of dry ice (0.5-1 kg)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hammer and hard flat object</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Two 5 L plastic containers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bands</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large flour sifter or sieve strainer- preferably one with a detachable plastic ring on the circumference</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Several large and small round bottom stainless steel containers. One of them should be larger than the flour sifter&#39;s circumference (approximately 40 cm in diameter), to minimize the loss of the sifted mass outside the round bottome stainless steel container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-chilled (via liquid nitrogen) stainless steel spoon, spatula, and scoopula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Several clothespins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-chilled (via liquid nitrogen) labeled 1.5 mL tubes with holes poked on the lid with a sterile needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Two containers of liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 cm wide painting brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

non-aqueous isolation and enrichment of glandular capitate stalked and sessile trichomes from cannabis sativa

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.

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 &#946;-mercaptoethanol) used in traditional trichome isolation medium, the work is not restricted to a chemical hood...

Watch this Scientific Journal Video about Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa at JoVE.com

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

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,&#160;dry ice, and nylon sieves and is suitable for RNA extraction and transcriptomic analysis.

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

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from ...

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