The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.
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.
Arabidopsis thaliana (Arabidopsis) is a prime model organism for basic and applied research in plant biology1,2,3. While standard conditions for Arabidopsis growth have been well established4, the effect of seed sterilization on seed viability has not been rigorously tested. Solid media in plates or boxes is routinely used to facilitate growth of Arabidopsis seedlings for many experimental applications, such as identification of homozygous lethal mutants in a segregating population, observation of shoot and root phenotypes at early stages, isolation of pathogen-free tissue, collection of large amounts of seedling tissue, selection of transformants or drug-resistant plants and evaluation of germination1,2,3,4. Seeds harvested from plants grown in a greenhouse or growth chamber are occasionally contaminated with microorganisms and dust. Growth of Arabidopsis seedlings on different types of sterile media requires prior seed sterilization to remove microbial contaminants such as fungi and bacteria present on the seed surface. The use of an effective seed sterilization regime is critical for a balance of high germination, minimum contamination, and vigorous plant growth.
The two most common methods used for Arabidopsis seed sterilization are based on commercial bleach (liquid-phase) and chlorine gas (vapor-phase). Various procedures have been employed for both liquid-phase sterilization1,4,5,6,7,8,9,10 and vapor-phase sterilization of Arabidopsis seeds8,10,11,12,13,14,15,16. However, while these procedures have been effective in accomplishing seed sterilization of the utilized genotypes, a detailed analysis of the effect of different sterilization treatments on the seeds of different genotypes has not been reported. Therefore, the optimization of these sterilization procedures is required to define conditions in which efficient sterilization is combined with high germination rate.
The Arabidopsis Biological Resource Center (ABRC) is uniquely positioned to a) test seed viability of a variety of different genotypes in the collection and b) take advantage of the quality control procedures applied internally and in response to user feedback about seed germination. The objectives of the experiments presented here were to determine the effects of various sterilization methods on seed germination of a range of Arabidopsis genotypes. Optimized sterilization procedures which result in high seed germination rates while maintaining minimal pathogen contamination are presented for both bleach and chlorine gas sterilization.
When growing Arabidopsis seeds on sterile media, some form of sterilization must be applied. Both bleach and chlorine gas sterilization treatments result in similar germination rate and mold growth inhibition. Neither sterilization method causes a significant reduction in germination rate for seeds with high germination potential; however, bleach sterilization is recommended for lines with lower germination potential (20-70%), due to the small, albeit non-significant, improvement in germination rate compared to gas sterilization (Figure 3A).
Sterilizing Arabidopsis seeds with bleach concentrations from 40-100% for up to 10 min provides satisfactory germination percentages and effective mold suppression. Although bleach concentrations less than 40% provide adequate sterilization for most seed lots, using a concentration of 40% or higher guarantees effective sterilization of even heavily contaminated seed lots. It is important not to exceed 10 min of sterilization when using bleach concentrations equal or higher than 80% to avoid high seed mortality and defects in seedling development.
Treating Arabidopsis seeds with chlorine gas concentrations of 6.1% or 16.5% for 1 h results in high germination rates and adequate mold elimination. Low chlorine gas concentration (2.1%) can be used successfully by increasing duration of sterilization to 3 h.
When few lines need to be sterilized, liquid sterilization in a solution of 50% bleach for 10 min is recommended. For greater numbers of lines, gas sterilization with a gas concentration of 6.1% for 1 h is a better option since many lines can be sterilized quickly and easily with less manipulation.
Our results provide standardized conditions for sterilizing both large number of seeds of different genotypes and the seeds with lower germination potential. The only limitation of these sterilization techniques is that they cannot be applied to the seeds with germination rates less than 20% due to extensive seed mortality. Alternative methods, such as sonication17, to increase germination rate in the absence of sterilization might be beneficial in those cases.
The authors have nothing to disclose.
We would like to thank Gauri Datta for her assistance in preparing experimental materials. We are also grateful to Bettina Wittler and James Mann for the critical review of the manuscript. This work was supported by the NSF grants DBI-1049341 and MCB-1143813.
0.65 mL Microcentrifuge tubes | GeneMate | C-3260-5 | Tube in which Arabidopsis thaliana seeds are placed to perform sterilization |
1.7 mL Microcentrifuge tube | GeneMate | C-3262-1 | Tube in which Arabidopsis thaliana seeds are placed to perform sterilization |
7 L plastic container | Sistema | 1016265438 | Container in which gas sterilization is performed |
Concentrated HCl | Sigma-Aldrich | 320331 | Chemical used in the process of creating chlorine gas |
Disposable sterile inoculating loop | Fisher Scientific | 22-363-603 | Loop is used to spread or position Arabidopsis seeds on MS plates |
Gamborg’s vitamin solution | Sigma-Aldrich | G1019 | Vitamin solution used in the process of making MS media |
Household bleach | Clorox | Regular-Bleach2 | Chemical used in the process of creating chlorine gas and liquid sterliziation |
MES hydrate | Sigma-Aldrich | M2933 | Chemical used in the process of making MS media |
Micropore surgical tape | 3M | 1530-1 | Microporous surgical paper tape used to seal MS plates |
Murashige and Skoog basal salt mixture (MS) | Sigma-Aldrich | M5524 | Chemical used in the process of making MS media |
Parafilm M | Bemis Company | #PM996 | Parraffin film used to seal sterilization container |
Petri dish 100 X 15 mm | Fisher Scientific | FB0875713 | Petri dishes in which MS media is poured for the purpose of growing Arabidopsis thaliana |
pH indicator strips | Whatman | 2613991 | Used to check pH of neutralizied chlorine and sodium bicarbonate solution |
Phytoagar | Fisher Scientific | 50-255-212 | Used to aid in the suspension of Arabidopsis seeds in the process of plating seeds |
Sodium bicarbonate | Sigma-Aldrich | S5761 | Chemical used in the process of neutralizing chlorine gas reaction |
Sucrose | Sigma-Aldrich | S0389 | Chemical used in the process of making MS media |
Tween 20 | Fisher BioReagents | BP337-100 | Chemical used in the process of liquid sterilization |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | CS70000 (Col-0) | Arabidopsis wild-type seeds |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_037606C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_041402C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_059101C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_063470C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_072048C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_072240C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081989C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_084124C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_085049C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_089717C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_107354C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110111C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111322C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113109C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_114702C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_114872C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_115657C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_116803C | Arabidopsis seeds used in testing as a part of Group 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_039445C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_039782C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_043037C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_045828C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_048556C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_049514C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_049725C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_080816C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081176C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081770C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_082262C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_082289C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_082702C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_083630C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_084635C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_085337C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_085656C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_093049C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_103332C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_105336C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_105704C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_106388C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_109575C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110580C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110617C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111424C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111584C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_112097C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113339C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_115837C | Arabidopsis seeds used in testing as a part of Group 2 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_019535C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_026478C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_046565C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_049258C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_049339C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_056307C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081292C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081597C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_083488C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110573C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_112793C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113658C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113904C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_114673C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_114709C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_115455C | Arabidopsis seeds used in testing as a part of Group 3 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_013186C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_018261C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_062509C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_080639C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_088586C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_096651C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_106900C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110131C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111051C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_111245C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_113223C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_121391C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_125097C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_201905C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_210001C | Arabidopsis seeds used in testing as a part of Group 4 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_000662C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_029335C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_047760C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_071275C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_080530C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_103881C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_110864C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_120294C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_124390C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_132808C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_137036C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_139519C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_140643C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_142288C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_143304C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_147597C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_209076C | Arabidopsis seeds used in testing as a part of Group 5 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_081081C | Arabidopsis seeds used in testing as a part of Groups 5 and 1 |
Arabidopsis thaliana seeds | ABRC; order through TAIR www.arabidopsis.org | SALK_107487C | Arabidopsis seeds used in testing as a part of Groups 5 and 2 |