Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Abstract

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

Inheritance of seed and rhizosphere microbial communities through plant–soil feedback and soil memory

“…However, seeds and flowers serve as the carriers of plant pathogens. Thus, over the past several decades, a variety of phytosanitary measures have been developed for seed sterilization (Miché and Balandreau, 2001;Godefroid et al, 2016;Lindsey et al, 2017). However, recent developments in multi-omics-based analytical techniques suggest that seeds contain plant genotype-specific microorganisms that can be transferred vertically from one generation to the next (Adam et al, 2018;Johnston-Monje et al, 2016;Nelson, 2017;Shade et al, 2017).…”

Antimicrobial solid media for screening non-sterile Arabidopsis thaliana seeds

“…When producing new A. thaliana transgenic lines, experimental throughput is partly limited by the transformant screening process. In particular, the seed sterilization step is time consuming and can cause seed mortality [7]. Alternative screening methods that negate the need for seed sterilization have been developed, each with advantages and disadvantages.…”

“…The seed sterilization step in particular has disadvantages that become more pronounced when screening increasing numbers of transformant lines. Liquid sterilization in hypochlorite bleach has a low seed mortality rate but is tedious, requiring several washing steps that become time consuming when preparing large quantities of seeds [7]. Chlorine gas is suitable for sterilizing seeds from multiple lines simultaneously, but gas sterilization still requires up to four hours of waiting time and can have a relatively high mortality rate even when the gas concentration is carefully controlled [7].…”

“…Liquid sterilization in hypochlorite bleach has a low seed mortality rate but is tedious, requiring several washing steps that become time consuming when preparing large quantities of seeds [7]. Chlorine gas is suitable for sterilizing seeds from multiple lines simultaneously, but gas sterilization still requires up to four hours of waiting time and can have a relatively high mortality rate even when the gas concentration is carefully controlled [7]. Mortality caused by the sterilization process could result in the loss of rare transformants or a reduction in the diversity of mutant libraries.…”

A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses

“…Arabidopsis thaliana accession Columbia-0 was used as the background for all amiRNA transformations of the library. Surface sterilized seeds (15 min 70% Ethanol, 0.1% Sodium dodecyl sulfate; 3-4 washes with ∼ 100% Ethanol; alternatively 10 min 50 % bleach, 0.05% Tween 20; 4 - 6 washes with water (Lindsey et al , 2017) of A. thaliana were cold-treated for 2 - 5 days at 4°C and germinated on half strength Murashige and Skoog basal medium supplemented with Gamborg’s vitamins (Sigma-Aldrich (Murashige and Skoog, 1962; Gamborg et al , 1968), 0.8% Phytoagar (Difco, Franklin Lakes, NJ) and pH adjusted (pH 5.8; 4-Morpholinoethane sulfonic acid (2.6 mM; Sigma-Aldrich) titrated with potassium hydroxide). After 5 – 7 days, plants were transferred to plastic pots containing sterilized premixed soil (Sunshine Professional Blend LC1 RS; Sunshine; supplemented with an appropriate amount of insecticide (Marathon, Gnatrol)) and propagated under the following conditions: long day (16hr light/ 8 hrs.…”