Pour the bleach into the developing tank and turn it upside down a few times to mix it in. Again, agitate the solution with the agitation stick every minute. Leave the bleach in for 3 to 3 1/2 minutes. Keep checking the temperature of the fixer while you wait and put the stabilizer in the washing tub to warm up.
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.
Bleach is the most commonly used agent for seed sterilization in numerous plant species. The optimal concentration of the sterilization agent and the exposure time varies between species. A number of protocols have been employed using bleach for sterilization of Arabidopsis seeds1,4,5,6,7,8,9,10. Four different concentrations of bleach with five different exposure time periods were tested and the results are presented inFigure 1. The treatments were applied to Columbia wild-type (Col-0) seeds. The effect of bleach concentration on germination of Col-0 seeds varied depending on the time of sterilization as demonstrated by a significant interaction between bleach concentration and time of sterilization (Figure 1, P < 0.001, ANOVA -Analysis of variance).
In the experiments with sterilization times between 5 and 10 min, treatments with all bleach concentrations resulted in equally high germination rate of Col-0 seeds (Figure 1). High germination rates were also observed for household bleach concentrations of 40% and 50% for all sterilization times. Treatments with 80% and 100% bleach for periods longer than 10 min resulted in a significant decrease in germination rate compared to the shorter soaking times (P < 0.01, ANOVA). Furthermore, for both 80% and 100% bleach treatments for 20 min, germination was significantly decreased compared to the corresponding 40% and 50% bleach treatments (P < 0.001, ANOVA).
Seeds displayed various levels of bleaching and shriveling when using high bleach concentrations for 15 min or longer. In addition to relatively high (up to 32%) seed mortality, germinated seeds sterilized in these conditions often showed growth defects, reflected in a failure of the cotyledons and hypocotyls to unfold and elongate, resulting in developmental arrest. Most treatments (14 out of 20) were completely mold-free, resulting in overall mold level average of 0.21% 0.003 (Table 1).
A treatment with 50% bleach and a soaking time of 10 min was selected as the best sterilization regime because it combined high germination percentage with good inhibition of surface pathogen growth. This treatment was selected to test the effect of bleach sterilization on different mutant lines as described below.
Statistical analysis showed that the germination response to sterilization methods was dependent on the germination potential of the lines (Figure 3A, P < 0.01, ANOVA). Neither bleach nor chlorine gas sterilization reduced germination rate of seeds with high germination potential (Groups 4 and 5). Neither treatment had an effect on the already low germination rate of the group with the lowest germination potential (Group 1). In contrast, chlorine gas sterilization resulted in a significant reduction by approximately 12-18% (P < 0.01, ANOVA) in the germination of seeds with intermediate germination potential (Groups 2 and 3). Bleach sterilization also decreased germination rate by 13% of Group 2, but it did not decrease the germination rate of Group 3. Although there was no significant difference in germination rate between the bleach and chlorine gas treatments in any germination group (Figure 3A, P > 0.442, ANOVA), seeds sterilized using bleach did have a slightly higher germination rate than gas-sterilized seeds in all germination groups.
Sterilization treatments significantly altered (P < 0.001, ANOVA) the percent of seeds affected by mold (Figure 3B). Both chlorine gas and bleach sterilization resulted in less mold growth (P < 0.05, ANOVA) compared to no sterilization. There was no difference in mold level detected between gas and bleach sterilization in any group (Figure 3B, P > 0.4, ANOVA).
Figure 1: Effects of bleach concentration and sterilization time on germination of Arabidopsis Col-0 seeds. Values are means SD obtained from 5 independent replications of the experiment. *indicates significant differences relative to the 5 min soaking duration for a range of bleach concentrations (P < 0.01, ANOVA). #indicates significant differences of 80% and 100% bleach concentrations relative to 40% and 50% for a 20 min time period (P < 0.01, ANOVA). Please click here to view a larger version of this figure.
Figure 3: Effects of bleach and chlorine gas sterilization on seeds with different germination potential. (A) Germination rate and (B) Mold level. The 100 SALK T-DNA lines were classified into five groups according to their germination potential defined as the germination rate in the absence of any sterilization agent. The groups based on the germination rate were as follows: Group 1 (0-20%), Group 2 (21-50%), Group 3 (51-70%), Group 4 (71-90%) and Group 5 (91-100%). The lines were chosen randomly and their germination potential did not depend on genotype. Values are means SD obtained from three independent replications of the experiment. Letters (\"a\", \"ab\", \"c\", etc.) above each value indicate the statistical groupings of category means. Means that do not share a letter are significantly different (P < 0.05, ANOVA). Please click here to view a larger version of this figure.
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.
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.
The X-T4 has 12 color simulation modes, including Acros, Classic Chrome, Classic Neg, Provia, and Velvia. The newcomer is Eterna Bleach Bypass which is different than Eterna, and as the name suggests, adds bleach bypass to the image. 59ce067264