High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Summary

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Abstract

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

Introduction

Arabidopsis is a diploid plant species belonging to the Brassicaceae family. Its relatively short life cycle (two months per generation under long-day growing conditions), small plant size, and self-pollination with the production of hundreds of seeds per plant have made it the first fundamental plant model species^1,2. In addition, its genome was fully sequenced³, extensive reverse genetics tools (saturated T-DNA, transposon, and chemically mutagenized populations) are available^4,5,6, and effec....

Protocol

1. Reagents and media preparation

1. Prepare 70% ethanol solution: Add 737 mL of 95% technical ethanol to 263 mL of distilled water. Mix thoroughly.
   NOTE: Prepare 70% ethanol solution on a non-sterile working bench.
   CAUTION: Ethanol is highly flammable and can cause serious irritation to the eyes. Keep away from flames and heat sources. In case of contact with eyes, rinse with abundant water.
2. Prepare 5% bleach solution: Add 5 mL of household bleach (containing ~3.5% of Sodium hypochlorite, NaClO) to 95 mL of sterile distilled water. Add a few drops of non-ionic detergent (e.g., Tween 20) and mix thoroughly.
   NOTE: Prepare 5% bleach sol....

Results

In order to assess the time required for the entire seed sterilization procedure, the time differences for liquid handling 96 samples in the current protocol were calculated and compared with traditional pipetting methods. The result indicates that the current protocol saves time, cutting the liquid handling time to one-fourth of that with the traditional protocols (Table 1). The table further highlights that the liquid removal time in the current protocol saves more time than that of the traditional met.......

Discussion

Sterilization of seeds is the fundamental step for functional studies in Arabidopsis. Although it is frequently carried out for many different purposes, limited studies on high-throughput seed surface sterilization in Arabidopsis are available.

So far, one of the methods with the highest throughput is using chlorine gas generated by mixing bleach with concentrated HCl. Although this method requires limited hands-on time, it uses a gas highly toxic to human beings²⁷. In .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aquarium valve	Amazon	B074CYC5SD	Kit including 2 valves and thin-walled tubings. The valve prevents the liquids to go back to the sterile tip
Arabidopsis Col-0 wild-type seeds	Nottingham Arabidopsis Stock Center	N1093	Wild type seeds (sensitive to kanamycin)
Arabidopsis transgenic line AdoIspS-79 seeds	NA	NA	Transgenic line overexpressing an isoprene synthase gene from Arundo donax transformed in the Col-0 background, resistant to kanamycin (Li et al. (2017) Mol. Biol. Evol., 34, 2583–2599). Available on request from the authors
Microcentrifuge	Eppendorf	EP022628188	Benchtop microcentrifuge used for spinning down the seeds
Murashige & Skoog medium including vitamins	Duchefa	M0222	Standard medium for plant sterile culture
Pipette controller	Brand	26300	Used to operate the serological pipette
Polyethylene tube 1	Roth	9591.1	Tube for connection from vacuum pump to decantation bottle (inner diameter: 7 mm; outer diameter: 9 mm)
Polyethylene tube 2	Roth	9587.1	Tube for connection from decantation bottle to the aquarium valve  (inner diameter: 5 mm; outer diameter: 7 mm)
Screw cap with connectors	Roth	PY86.1	2-way dispenser screw cap GL45 in polypropylene for decanting bottle
Serological pipette	Brand	27823	Graduated glass (reusable) serological pipette. Disposable pipettes can be used instead
Shakeret al.	Qiagen	85300	TissueLyser II bead mill used normally for tissue homogenization. Without the addition of beads to the tubes it works as shaker.
Technical ethanol	ITW Reagents (Nova Chimica Srl)	212800	Ethanol 96% v/v partially denatured technical grade
Tween 20	Merck Millipore	655205	Non-ionic detergent acting as surfactant
Universal tubing connectors	Roth	Y523.1	Can be used to improve/simplify tubing connections
Vacuum pump	Merck Millipore	WP6222050	Used for making the suction device