Production location of the gelling agent Phytagel has a significant impact on Arabidopsis thaliana seedling phenotypic analysis.

BACKGROUND: Recently, it was found that 1% Phytagel plates used to conduct Arabidopsis thaliana seedling phenotypic analysis no longer reproduced previously published results. This Phytagel, which is produced in China (Phytagel C), has replace American-made Phytagel (Phytagel), which is no longer commercially available. In this study, we present the impact of Phytagel produced in the United States vs. China on seedling phenotypic analysis. As a part of this study, an alternative gelling agent has been identified that is capable of reproducing previously published seedling morphometrics.
RESULTS: Phytagel and Phytagel C were investigated based on their ability to reproduce the subtle phenotype of the sob3-4 esc-8 double mutant. Fluence-rate-response analysis of seedlings grown on 1% Phytagel C plates failed to replicate the sob3-4 esc-8 subtle phenotype seen on 1% Phytagel. Furthermore, root penetrance analysis showed a significant difference between sob3-4 esc-8 seedlings grown on 1% Phytagel and 1% Phytagel C. It was also found that 1% Phytagel C was significantly harder than 1% Phytagel. As a replacement for Phytagel C, Gellan was tested. 1% Gellan was able to reproduce the subtle phenotype of sob3-4 esc-8. Furthermore, there was no significant difference in root penetration of the wild type or sob3-4 esc-8 seedlings between 1% Phytagel and 1% Gellan. This may be due to the significant reduction in hardness in 1% Gellan plates compared to 1% Phytagel plates. Finally, we tested additional concentrations of Gellan and found that seedlings on 0.6% Gellan looked more uniform while also being able to reproduce previously published results.
CONCLUSIONS: Phytagel has been the standard gelling agent for several studies involving the characterization of subtle seedling phenotypes. After production was moved to China, Phytagel C was no longer capable of reproducing these previously published results. An alternative gelling agent, Gellan, was able to reproduce previously published seedling phenotypes at both 1% and 0.6% concentrations. The information provided in this manuscript is beneficial to the scientific community as whole, specifically phenomics labs, as it details key problematic differences between gelling agents that should be performing identically (Phytagel and Phytagel C).

Background

The study of phenomics in Arabidopsis thaliana (A. thaliana) is the focus of many molecular and physiology labs worldwide. One of the ways that A. thaliana growth and development can be studied is through the use of growth media plates. The use of growth media plates for the study of A. thaliana has many benefits, including affordability, transparency, ease, and most importantly, reproducibility.

Growth media plates are made with agar derived from red algae, or more commonly, by agar substitutes. One common agar substitutes is Phytagel (Sigma). Phytagel is produced from a bacterial substrate that is composed of rhamnose, glucuronic acid, and glucose [1]. Phytagel creates a clear, colorless growth matrix for plants. Another widely used agar substitute is gellan gum. Gellan Gum (Gellan) (PhytoTechnology Laboratories, Inc.) is produced by bacterial fermentation of Sphingomonas elodea, which creates a high molecular weight polysaccharide gum. This gum is composed of repeating tetrasaccahride units that will form a gel in the presence of mono- or divalent cations [2].

Growth media plates allow for the seeds of small plants, such as A. thaliana, to be grown and phenotyped in controlled environments. These plates are especially important when studying A. thaliana plants with subtle phenotypes. An activation tagging screen was conducted to identify suppressors of the long hypocotyl phenotype conferred by the weak, missense phyB-4 mutant allele [3-9]. From the activation tagging screen, SUPPRESSOR OF PHYTOCHROME B-4 #3 (SOB3) and its closest paralog, ESCAROLA (ESC), were identified [3–4, 9]. Null alleles of both SOB3 and ESC were identified as sob3-4 and esc-8, respectively [9]. It was observed that the sob3-4 esc-8 double mutant produced a subtle hypocotyl phenotype that was taller than the wild type (WT), but shorter than the extreme-tall phenotype of the dominant-negative sob3-6 allele [9,10].

1% Phytagel plates containing 1.5% sucrose were used as a standard to conduct all of the aforementioned phenomics research. Additionally, 1% Phytagel plates have been used to reproduce the results of the sob3-6 mutant [11], as well as in new research with the subtle phenotypes of A. thaliana NAC Domain Containing Protein 81 (ATAF2) mutants [12]. Therefore, 1% Phytagel plates are sufficient for phenotypic analysis of subtle mutant phenotypes and the results are reproducible.

In 2016, we discovered issues with our standard growth media plates. The plates were harder to the touch, the seedlings appeared to be germinating asynchronously, and did not look healthy. After notifying Sigma of these issues and receiving Phytagel from a different lot number, the problems persisted. We soon discovered that both of the new batches of Phytagel were now being produced in China (Phytagel C). We compared growth of A. thaliana seedlings on plates made with American-made Phytagel (Phytagel) to their growth on plates made with Phytagel C. We included another gelling agent, Gellan, produced by PhytoTechnology Laboratories Inc., in our experimentation. The purpose of these experiments was to explore how different gelling agents performed under light intensities commonly used for seedlings phenomics, and to possibly indicate the gelling agent that produced the most uniform germination, root penetrance, and/or hypocotyl fluence rate responses. In this study we present the impact of Phytagel produced in the United States vs. China on seedling phenotypic analysis. As a part of this study, an alternative gelling agent has been identified that is capable of reproducing previously published seedling morphometrics.

Results

Hypocotyl length measurements, as well as fluence-rate-response analysis, have been used to elucidate the subtle mutant phenotypes, such as phyB-4 [3] and sob3-4 esc-8 [9]. The standard for these experiments is 1% Phytagel media [3,9,11]. The differences between these subtle phenotypes can be best observed at a white light intensity of 10 μmol m-2s-1 (Fig 1; Fig 2, 2A and 2C) [9]. The sob3-4 esc-8 double mutant has been observed to be significantly taller than the WT, but shorter than sob3-6 [9]. This difference in average hypocotyl length between WT and sob3-4 esc-8 on 1% American-made Phytagel is statistically significant (Fig 2 and 2B), but this difference is not statistically significant on 1% Phytagel C media (Fig 2 and 2D). However, both Phytagel and Phytagel C are able to distinguish the difference between the WT and more severe mutant phenotypes conferred by SOB3-D and sob3-6 (Fig 2, 2B and 2D). Interestingly, WT dark grown seedlings on Phytagel and Phytagel C media are significantly different, but no such significance is observed for dark grown sob3-4 esc-8 seedlings on Phytagel and Phytagel C media (see S2 Fig). In addition, the length of the hypocotyls for all genotypes are significantly different (at least p < 0.01) between 1% Phytagel C media and 1% Phytagel media (Fig 2, 2B and 2D). This led us to suspect that there may be an issue with seedling germination or seedling development on Phytagel C media. Therefore, we conducted germination and root penetrance assays to determine if either of these factors are impacted by Phytagel C media.

Fig 1

Pictures of seedlings grown on 1% media at 10 μmol m-2s-1 for six days.

A) 6-day-old seedlings grown on 1% Phytagel at 10 μmol m-2s-1. B) 6-day-old seedlings grown on 1% Phytagel C at 10 μmol m-2s-1.

Fig 2

Fluence rate responses of 6-day-old seedlings on 1% Phytagel and 1% Phytagel C plates.

A) Fluence rate responses of 6-day-old seedlings that have been normalized to the dark control on plates containing 1% Phytagel media. B) Graphical representation of 6-day-old seedlings that have been normalized to the dark control on plates containing 1% Phytagel media at 10 μmol m-2s-1. C) Fluence rate responses of 6-day-old seedlings that have been normalized to the dark control on plates containing 1% Phytagel C media. D) Graphical representation of 6-day-old seedlings that have been normalized to the dark control on plates containing 1% Phytagel C media at 10 μmol m-2s-1. Standard error is shown for all data sets. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with the wild type: P > 0.05 = NS, P ≤ 0.0001 = ****.

Germination rates were calculated for each of the four genotypes (WT, sob3-6, SOB3-D, and sob3-4 esc-8) on 1% Phytagel and 1% Phytagel C plates at different light intensities (10, 60, and 100 μmol m-2 s-1). No trends were observed that would indicate a clear connection between gelling agent and percent germination (see S1 Fig). However, root penetrance was impacted by growth on 1% Phytagel C media. It was found that there is no significant difference between Phytagel and Phytagel C at 10 and 100 μmol m-2s-1 for the WT, but there is a significant difference at 60 μmol m-2s-1 (Fig 3 and 3A). Furthermore, the average percent root penetrance for sob3-4 esc-8 at all light intensities were statistically different between 1% Phytagel and 1% Phytagel C plates (Fig 3 and 3B), suggesting that there is physical difference between these two gelling agents. In order to test if the Phytagel was physically different from Phytagel C, a hardness analysis was performed (Fig 4). The 1% Phytagel C plates were significantly harder than the 1% Phytagel plates (Fig 4), which may explain the root penetrance data in Fig 3.

Fig 3

Average root penetrance of 6-day-old seedlings on 1% Phytagel and 1% Phytagel C plates.

A) Average root penetrance of 6-day-old WT seedlings on Phytagel and Phytagel C at increasing light concentrations. B) Average root penetrance of 6-day-old sob3-4 esc-8 seedlings on Phytagel and Phytagel C at increasing light concentrations. Standard error is shown for all data sets. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with Phytagel: P > 0.05 = Not Significant (NS), P ≤ 0.01 = **, and P ≤ 0.001 = ***.

Fig 4

Force test of 1% Phytagel and 1% Phytagel C plates.

Force required to penetrate a centimeter of 1% growth media containing either Phytagel or Phytagel C as the gelling agent. Three plates for each gelling agent were prepared identically. 10 samples were taken from each plate and averaged. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with Phytagel: P ≤ 0.0001 = ****.

Since the original Phytagel is no longer available for purchase, and Phytagel C has an adverse impact on seedling phenomics, another gelling agent, Gellan, was compared to Phytagel. Gellan was able to distinguish the subtle phenotype of sob3-4 esc-8 through fluence-rate-response analysis (Fig 5, 5A and 5C) and was able to significantly separate sob3-4 esc-8 from the WT (Fig 5, 5B and 5D).

Fig 5

Fluence rate responses of 6-day-old seedlings on 1% Phytagel and 1% Gellan plates.

A) Fluence rate responses of 6-day-old seedlings that have been normalized to the dark control on plates containing 1% Phytagel media. B) Graphical representation of 6-day-old seedlings that have been normalized to the dark control on plates containing 1% Phytagel media at 10 μmol m-2s-1. C) Fluence rate responses of 6-day-old seedlings that have been normalized to the dark control on plates containing 1% Gellan media. D) Graphical representation of 6-day-old seedlings that have been normalized to the dark control on plates containing 1% Gellan media at 10 μmol m-2s-1. Standard error is shown for all data sets. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with the wild type, P ≤ 0.0001 = ****.

Percent root penetrance on 1% Gellan plates were not negatively impacted for any of the genotypes at any light fluence-rate when compared to 1% Phytagel plates (Fig 6 and 6A). In addition, in all conditions the percentage of root penetrance on 1% Gellan plates was higher than on 1% Phytagel plates (Fig 6 and 6B). The root penetrance data can be explained, at least in part by, the observation that 1% Gellan plates are softer than 1% Phytagel plates (Fig 7). This may also explain the visual difference we see between 1% Phytagel C and 1% Gellan plates (Fig 8).

Fig 6

Average root penetrance of 6-day-old seedlings on 1% Phytagel and 1% Gellan plates.

A) Average root penetrance of 6-day-old WT seedlings on Phytagel and Gellan at increasing light concentrations. B) Average root penetrance of 6-day-old sob3-4 esc-8 seedlings on Phytagel and Gellan at increasing light concentrations. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with Phytagel: P > 0.05 = Not Significant (NS), P ≤ 0.05 = *.

Fig 7

Force test of 1% Phytagel and 1% Gellan plates.

Force required to penetrate a centimeter of 1% growth media containing either Phytagel or Gellan as the gelling agent. Three plates for each gelling agent were prepared identically. 10 samples were taken from each plate and averaged. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with Phytagel: P ≤ 0.0001 = ****.

Fig 8

Photos of seedlings grown on 1% media at 10 μmol m-2s-1 for six days.

A) 6-day-old seedlings grown on 1% Gellan at 10 μmol m-2s-1. B) 6-day-old seedlings grown on 1% Phytagel C at 10 μmol m-2s-1.

Since Phytagel is no longer available and Phytagel C is not a viable alternative, a replacement gelling agent needed to be identified. Since 1% Gellan has been shown to reproduce previously published results (Figs 5 and 6), we tested various concentrations to determine a new standard for the lab. 0.6% Gellan plates were able to reproduce the same previously published results (Figs 9 and 10). We replicated the 0.6% experiment with Phytagel C (Fig 11). Even at this lower concentration, Phytagel C was not able to distinguish the sob3-4 esc-8 phenotype from the WT phenotype.

Fig 9

Photos of 6-day-old seedlings grown on media at 10 μmol m-2s-1.

A) 6-day-old seedlings grown on 0.6% Gellan at 10 μmol m-2s-1. B) 6-day-old seedlings grown on 1% Gellan at 10 μmol m-2s-1.

Fig 10

Fluence rate responses of 6-day-old seedlings plates containing 0.6% Gellan media.

A) Fluence rate responses of 6-day-old seedlings that have been normalized to the dark control on plates containing 0.6% Gellan media. B) Graphical representation of 6-day-old seedlings that have been normalized to the dark control on plates containing 0.6% Gellan media at 10 μmol m-2s-1. Standard error is shown for all data sets. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with the wild type, P ≤ 0.001 = *** and P ≤ 0.0001 = ****.

Fig 11

Fluence rate responses of 6-day-old seedlings on plates containing 0.6% Phytagel C media.

A) Fluence rate responses of 6-day-old seedlings that have been normalized to the dark control on plates containing 0.6% Phytagel C media. B) Graphical representation of 6-day-old seedlings that have been normalized to the dark control on plates containing 0.6% Phytagel C media at 10 μmol m-2s-1. Standard error is shown for all data sets. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with the wild type, P > 0.05 = Not Significant (NS), P ≤ 0.0001 = ****.

Discussion

Seedling phenomics is an important area of research that relies on the reproducibility of growth media plates. This is especially important when seedlings display subtle phenotypes, as in the case of the A. thaliana missense allele, phyB-4, and the double mutant, sob3-4 esc-8. In our study, we aimed to uncover roles that different gelling agents could be playing in A. thaliana seedling growth and development. Phytagel produced in America has been used for several studies involving the characterization of subtle seedling phenotypes [3,9,11]. Phytagel produced in China does not replicate these results in at least three distinct ways: fluence-rate-response analyses, root penetrance analyses, and hardness assays.

The disparity in hardness between the 1% Phytagel and 1% Phytagel C plates may explain the difference we see in seedling growth. For example, the hardness of the 1% Phytagel C plates may not be impacting the ability of the seeds to germinate, but it may be impacting the ability of the roots to penetrate the media and allow for proper growth. This may explain why we do not see an impact on germination rates for sob3-4 esc-8, but we saw a significant impact on the ability of the sob3-4 esc-8 roots to penetrate. We suspect that the hardness of the plate may be due to a chemical change in the Phytagel C media when exposed to light. The change in seedling growth and development could also be due to a change in water potential within the media. Further testing is needed to determine the cause of the increased hardness of the Phytagel C plates.

Since Phytagel C was not able to give reproducible results, we tested another gelling agent, Gellan, to determine if it was a suitable replacement for Phytagel. We suspected that the cause for similar root penetrance between 1% Gellan and 1% Phytagel plates may be due to likeness in hardness. However, we found that 1% Gellan plates are significantly less hard than 1% Phytagel plates. Chemical and/or osmotic experimentation would possibly clarify the differences in hardness that is seen between the three different gelling agents.

After the aforementioned experiments, the Neff lab decided to replace Phytagel with Gellan for phenotypic experimentation on A. thaliana seedlings. This was a necessary replacement, as seedling phenomics were halted in the Neff lab without a reliable gelling agent. Since the 1% protocol had been established with Phytagel, we tested different concentrations of Gellan to establish a new standard. We found that 0.6% Gellan plates gave more uniform visual results than 1%, reproduced previously published results, and is a more cost-effective option. Therefore, the Neff lab has replaced 1% Phytagel with 0.6% Gellan for phenotypic analysis of v seedlings.

This study highlights key development differences of A. thaliana seedlings on different gelling agents. It doesn’t, however, explore the chemical or physical differences that may be causing the changes in growth for plants germinated on Phytagel compared to Phytagel C. Further analyses, such as water potential and metal composition, are needed to determine the cause of the changes in Phytagel presented within this manuscript. Additionally, we have not looked at the impact of Phytagel C on tissue culture assays and adult morphologies. Although this information may be of interest to those involved in the production of gelling agents, the main purpose of this study is to report the problems that we have encountered while also providing a viable replacement for seedling morphometric analysis.

Conclusion

Three gelling agents, Phytagel (no longer commercially available), Phytagel C, and Gellan, were investigated based on their ability to reproduce the subtle phenotype of the sob3-4 esc-8 double mutant. Fluence-rate-response analysis of 1% Phytagel C plates failed to replicate the sob3-4 esc-8 subtle seedling phenotype seen on 1% Phytagel. Furthermore, root penetrance analysis showed a significant difference in root penetration between sob3-4 esc-8 seedlings grown on 1% Phytagel and 1% Phytagel C. Finally, it was found that 1% Phytagel C was significantly harder than 1% Phytagel, which may be causing decreased sob3-4 esc-8 root penetrance, as well as affecting seedling growth and development.

As a substitute for Phytagel C, 1% Gellan was able to reproduce the subtle phenotype of the sob3-4 esc-8 double mutant. Furthermore, there was no significant difference in root penetration of the WT or sob3-4 esc-8 seedlings between 1% Phytagel and 1% Gellan. It was also found that 1% Gellan plates are significantly softer than the 1% Phytagel plates. These observations suggest that Gellan is a suitable replacement for Phytagel. In order to establish a new standard for the lab, we tested different percentages of Gellan media. We found that 0.6% Gellan also reproduces previously published phenotypes and is more cost effective.

The information provided in this manuscript is beneficial to the scientific community as whole, specifically phenomics labs, as it details key problematic differences between gelling agents that should be performing identically (Phytagel and Phytagel C). We also provide labs with additional information on a gelling agent, Gellan, which can replace the use of the no-longer commercially available Phytagel. These data will help to promote consistency of methodologies for better integration of data from different laboratories.

Methods

Growth media plates

50mL plates were made with media containing one-half-strength Linsmaier and Skoog modified basal media, 1.5% sucrose (m/v), and the appropriate amount (m/v) of gelling agent. The gelling agents used in this study are: Phytagel (Sigma), Phytagel C (Sigma), and Gellan (PhytoTechnology Laboratories). Experiments were conducted on growth media plates containing 0.6% or 1% of these gelling agents. The lot number for Phytagel is SLBJ1281V. The lot number for Phytagel produced in China is SLBQ4373V. This was the second batch of product and it was received on October 20, 2016. The first batch was received on July 21, 2016 (lot number SLBQ4319V). The lot number for Gellan is SUS0434045A. Sigma and PhytoTechnology Laboratories do not print the date of production nor the expiration date on their productions.

Experimental design

15 seeds with known, published phenotypes (WT, sob3-4 esc-8, sob3-6, and SOB3-D) were hand-plated onto evenly divided sectors on each plate. These seeds had been previously surface-sterilized, as described below, and were used for up to six weeks post-sterilization. The plates were kept in the dark at 4° Celsius for three days to synchronize germination. Post cold and dark treatment, the plates underwent a 12-hour red-light treatment in a growth chamber at 25° Celsius. After this red-light treatment, one plate from each gelling agent group was subjected to one of these four light treatments for a total of six days: dark, 10 μmol m-2s-1, 60 μmol m-2s-1, and 100 μmol m-2s-1. At the end of six days, each seedling was analyzed for root penetrance and germination. Root penetrance and germination were recorded separately as binary results: 1 for yes it penetrated/germinated and 0 for no it did not penetrate/germinate. Germination was defined by whether or not there was appearance of the root. For example, a seed that germinated, but did not penetrate the media was recorded as 1/0. After these data were recorded, the seedlings were transferred to transparencies. The transparencies were scanned to the computer and the hypocotyl length of each seedling was measured using ImageJ Software, and is described in detail below. To account for differences in timing of germination, seedling measurements were normalized to the average hypocotyl length of each specific genotype grown in the dark. Eleven replicates were conducted for each light treatment and gelling agent combination at 1% concentration. Seven replicates were conducted for each light treatment and gelling agent combination at 0.6% concentration.

Seed sterilization

For sterilization, seeds were placed in microcentrifuge tubes and covered with 75% alcohol containing 0.5% Triton X-100 (v/v) and placed on a shaker for 15 minutes. The liquid was pipetted off and the seeds were covered with 95% alcohol containing 0.5% Triton X-100 (v/v) and shaken for 10 minutes. The liquid was pipetted off and the seeds were covered with 95% alcohol (v/v) and shaken for five minutes. The liquid was pipetting off and the seeds were placed on sterilized filter paper and left to dry in a laminar airflow hood.

Chamber setup

The light chamber used is E30B (Percival Scientific, Inc.). The different light intensities were achieved through the use of mesh screens. The light intensities were measured before experimentation using a LI-250A mobile spectrophotometer (LI-COR Biosciences). The spectrophotometer was place in the center of the light chamber with the door closed. Periodic measurements were taken to ensure the light intensities were not fluctuating.

Measuring hypocotyl length via NIH ImageJ software

The transparencies were digitized with a flatbed scanner at 720 dpi. The transparencies included a ruler for measuring a 1mm length to set the parameters for measurements in ImageJ (The NIH). A length of 1mm was established in pixels for each image. The hypocotyls were measured from the top of hypocotyl to the beginning of the roots using the segmented line tool. The same researcher measured all of hypocotyls to ensure no discrepancies would occur in measuring the hypocotyls. The measurements were transferred to an Excel spreadsheet for analysis.

Hardness testing via FTA probe

A fruit texture analyzer probe (GS-14 Fruit Texture Analyzer, GÜSS Instruments, South Africa) was used to test the force required to penetrate 1cM of media. Ten locations were selected on each plate and tested. Three plates were made at 1% for Phytagel, Phytagel C, and Gellan. The values for like plates were averaged.

Analysis of data and statistics

Standard error was conducted on all applicable data sets and are included as error bars where appropriate. Welch’s t test (unpaired two-tailed t test with unequal variance) was also conducted where appropriate. P values are included as follows: P > 0.05 = Not Significant (NS), P ≤ 0.01 = **, P ≤ 0.001 = ***, and P ≤ 0.0001 = ****.

Germination rates on 1% Phytagel and 1% Phytagel C media.

A) Germination rates of WT, sob3-6, SOB3-D, and sob3-4 esc-8 at 10 μmol m-2s-1 on 1% Phytagel and 1% Phytagel C plates. B) Germination rates of WT, sob3-6, SOB3-D, and sob3-4 esc-8 at 60 μmol m-2s-1 on 1% Phytagel and Phytagel C plates. C) Germination rates of WT, sob3-6, SOB3-D, and sob3-4 esc-8 at 100 μmol m-2s-1 on 1% Phytagel and Phytagel C plates. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with the wild type, P > 0.05 = Not Significant (NS).

(TIFF)

Click here for additional data file.

Dark grown seedlings on 1% Phytagel, 1% Phytagel C, and 1% Gellan.

A) Hypocotyl lengths of WT seedlings on different growth media plates. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared Phytagel, P ≤ 0.01 = **, P > 0.05 Not Significant (NS). B) Hypocotyl lengths of sob3-4 esc-8 seedlings on different growth media plates. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared Phytagel, P ≤ 0.0001 = ****, P > 0.05 = Not Significant (NS). C) Hypocotyl lengths of sob3-6 seedlings on different growth media plates. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared Phytagel, P > 0.05 = Not Significant (NS). D) Hypocotyl lengths of SOB3-D seedlings on different growth media plates. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared Phytagel, P > 0.05 = Not Significant (NS).

(TIFF)

Click here for additional data file.

10 Mar 2020

PONE-D-20-01374

Production location of the gelling agent Phytagel has a significant impact on Arabidopsis thaliana seedling phenotypic analysis

PLOS ONE

Dear Dr. Neff,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Apr 24 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Robert Hoehndorf, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The manuscript has been reviewed by two experts in the field, and while they find the manuscript and findings of value, they have raised several concerns that should be addressed in a revision.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2)  Our internal editors have looked over your manuscript and determined that it is within the scope of our Plant Phenomics & Precision Agriculture Call for Papers. This collection of papers is headed by a team of Guest Editors for PLOS ONE. The Collection will encompass a diverse range of research articles spanning disciplines, methods and applications.  Additional information can be found on our announcement page: https://plos.io/phenomics.

If you would like your manuscript to be considered for this collection, please let us know in your cover letter and we will ensure that your paper is treated as if you were responding to this call. If you would prefer to remove your manuscript from collection consideration, please specify this in the cover letter.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors discovered a difference in performance of phenotyping experiments conducted using Phytagel produced in USA vs. the newest Phytagel produced in China. The experiments done were thorough and well presented.

Line 100: Please indicate a more accurate date of change in the production location.

Line 238: Did you test the effects of Phytagel C in reproducing other phenotypes not related to light? Is this difference only upon the application of light treatment?

METHODS:

-Missing the description of the method to measure fluence rate response

Lines 287- 292: Do not call them Agar (or agarose) plates, since you are using agar substitutes, call them growth media plates.

Lines 290-291: Please include year of manufacture and/or expliration date.

Could the effects be due to the use of old gelling agent (Phytagel)?

FIGURES:

Overall, I recommend you use box plots instead of bar plots, so you can identify possible outliers and display more information about your results.

Your results are quite impressive, have you shared them with Sigma company? If so, what do they suggest that could be the cause?

Reviewer #2: This Ms from the Neff lab compares the gelling agent used for phenotypic analyses of Arabidopsis thaliana seedlings. As the company that produces Phytagel changed their provider, the gelling agent changed in its characteristics. As this can strongly influence results and lead to the fact that results are no longer comparable this is a very important finding.

The authors used a very sensitive mutant as a marker to test different gelling agents and tested for several responses, hypocotyl elongation under different light conditions, germination rates and root penetrance.

The authors concluded that mainly the hardness of the media was responsible for the differences observed between the different gelling agents and tested an alternative that can reproduce previous findings.

The paper is well written, the experiments done with care and statistically evaluated.

To make this study more interesting for a larger audience I am missing that other parameters were not checked. Osmotic behavior of the gelling agents and/or different metal ions could have a big impact on the growth of seedlings, especially on the root penetration phenotype. Yet no studies were conducted in this regard - other sensitive mutants could have been included to rule out further problems with the media. Additionally, I am surprised to see that 1,5% sucrose was include in the media, especially for the hypocotyl elongation. Most often for light experiments no sucrose is included in the media. Residual sugars in the media could also have a big impact on the hypocotyl elongation and the overall growth performance. Did the authors see differences in the growth behavior on media without any added sugars?

Hypocotyl length was normalized to the dark control, but I did not find any data if the hypocotyl length in darkness varied under the different media.

As both germination and root penetrance were scored the authors should define more precisely how they scored germination.

In principle this study shows how differences in results between different labs could be explained, and as the phenotypes of the sub3-4 esc-8 double mutant were only detectable on the softer gelling agents phenotypes of other mutants might be more prominent on the harder gelling agent. Therefore the methods and materials section in papers should be precise and papers such as this one are helpful to be aware of changes made by the suppliers.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Apr 2020

Response to Reviewers

This is a resubmission of the manuscript “Production location of the gelling agent Phytagel has a significant impact on Arabidopsis thaliana seedling phenotypic analysis”. We’ve addressed all of the reviewers’ comments to the best of our ability. We hope that you now find this manuscript sufficient for publication in PLoS One. Below you will find a detailed explanation addressing each of the reviewers’ points.

Reviewer 1:

The authors discovered a difference in performance of phenotyping experiments conducted using Phytagel produced in USA vs. the newest Phytagel produced in China. The experiments done were thorough and well presented.

Authors’ response: Thank you for your comment. We tried to organize the data in the manuscript as efficiently as possible.

Line 100: Please indicate a more accurate date of change in the production location.

Authors’ response: We have reached out to Sigma to determine when the production of Phytagel was moved from the United States to China. The only answer we have received is that manufacturing of Phytagel moved to China in May of 2019. We informed Sigma that we had received two different batches of product produced in China from late 2016. We didn’t receive a response after multiple follow-up attempts. We’ve further clarified when we noticed these changes within the manuscript on lines 110-114. Additionally, the lot numbers for each gelling agent and the dates we received Phytagel C are now on lines 325-329.

Line 238: Did you test the effects of Phytagel C in reproducing other phenotypes not related to light? Is this difference only upon the application of light treatment?

Authors’ response: Thank you for your question. We did not test the effects of Phytagel C on phenotypes that are not related to light, as all of the genotypes that we work with in our lab are involved in light-mediated pathways. This manuscript is intended to be used a guide to other labs by outlining our issues with this gelling agent. Further research is needed to determine the scope of Phytagel C’s effects. This has been further clarified in the discussion on lines 287-289. Additionally, we have included a new supplemental figure (S2) detailing the differences between dark grown seedlings on different 1% media. This information has been added to the results section of the manuscript on lines 139-142.

Methods

Missing description of the method to measure fluence rate response

Authors’ response: Thank you for bringing our attention to this error. How we measured fluence rate response has now been appropriately updated within the manuscript on lines 365-367.

Lines 287-292: Do not call them agar or agarose plates, since you are using agar substitutes, call the growth media plates

Authors’ response: Thank you for your suggestion. We agree with you and have updated the manuscript to reflect this change.

Lines 290-291 Please include year of manufacture and or/expiration date

Authors’ response: The lot numbers have been added to the method section for all gelling agents, as well as the dates we received Phytagel C. Please see lines 325-329.

Could the effects be due to the use of old gelling agent (Phytagel):

Authors’ response: Thank you for your question. The Phytagel C used for our experiments was from a new container of gelling agent received at the end of 2016. We notified Sigma of the issue and they sent us a new container from a different lot number. The issues persisted, which is when we designed our experiments. We have further clarified this within the manuscript to make it clearer to the reader both in the background on lines 110-114 and in the methods section on lines 325-329.

Figures

Overall, I recommend you use box plots instead of bar plots, so you can identify possible outliers and display more information about your results.

Authors’ response: Thank you for your suggestion. We agree and have updated all bar graphs to box plots.

Your results are quite impressive, have you shared them with Sigma company? If so, what do they suggest that could be the cause?

Authors’ response: Thank you for your question. Please see the response to the question of old Phytagel above. In summary, we did not receive any suggestion or clarification from Sigma about what could be the cause of our issues.

Reviewer 2

This Ms from the Neff lab compares the gelling agent used for phenotypic analyses of Arabidopsis thaliana seedlings. As the company that produces Phytagel changed their provider, the gelling agent changed in its characteristics. As this can strongly influence results and lead to the fact that results are no longer comparable this is a very important finding. The authors used a very sensitive mutant as a marker to test different gelling agents and tested for several responses, hypocotyl elongation under different light conditions, germination rates and root penetrance. The authors concluded that mainly the hardness of the media was responsible for the differences observed between the different gelling agents and tested an alternative that can reproduce previous findings. The paper is well written, the experiments done with care and statistically evaluated.

Authors’ response: Thank you for your comments. We took great care in designing and performing the experiments detailed within this manuscript. We are pleased to hear that our data and writing has been well received. We agree that the information provided within this manuscript is important for the scientific community.

To make this study more interesting for a larger audience I am missing that other parameters were not checked. Osmotic behavior of the gelling agents and/or different metal ions could have a big impact on the growth of seedlings, especially on the root penetration phenotype. Yet no studies were conducted in this regard - other sensitive mutants could have been included to rule out further problems with the media.

Authors’ response: Thank you for your question. The purpose of this manuscript is to outline the problems our lab experienced with Phytagel now produced in China. In doing so, we are notifying the scientific community that their research may be affected by this new formula. This manuscript was not intended to fully describe the reasons behind our findings. We think it is important that this information be received by the community as soon as possible. We have further detailed this explanation in our discussion section on lines 287-289.

Additionally, I am surprised to see that 1,5% sucrose was included in the media, especially for the hypocotyl elongation. Most often for light experiments no sucrose is included in the media. Residual sugars in the media could also have a big impact on the hypocotyl elongation and the overall growth performance. Did the authors see differences in the growth behavior on media without any added sugars?

Authors’ response: Thank you for your inquiry. We stated within the manuscript that our lab has used a 1% Phytagel media as a standard. However, it wasn’t clear that this standard includes 1.5% sucrose. The mutants used within the manuscript were previously characterized using media that contained 1.5% sucrose. Therefore, we felt it was appropriate to continue to use the same media formula. We have clarified that 1.5% sucrose was part of the previously established 1% Phytagel standard in the background of the manuscript on line 104.

Hypocotyl length was normalized to the dark control, but I did not find any data if the hypocotyl length in darkness varied under the different media.

Authors’ response: Thank you for your comment. We have added a supplemental figure (S2) detailing the findings of the dark grown seedlings on the different media. This has been added to the results section of the manuscript on lines 139-142.

As both germination and root penetrance were scored the authors should define more precisely how they scored germination.

Authors’ response: Thank you for your comment. We have updated this information within the manuscript on line 346.

In principle this study shows how differences in results between different labs could be explained, and as the phenotypes of the sub3-4 esc-8 double mutant was only detectable on the softer gelling agents phenotypes of other mutants might be more prominent on the harder gelling agent. Therefore, the methods and materials section in papers should be precise and papers such as this one are helpful to be aware of changes made by the suppliers.

Authors’ response: Thank you for your comment. We agree, and we hope our changes to the manuscript allow for a more precise explanation of our methodology and findings.

Submitted filename: Repsonse to Reviewers.docx

Click here for additional data file.

29 Apr 2020

Production location of the gelling agent Phytagel has a significant impact on Arabidopsis thaliana seedling phenotypic analysis

PONE-D-20-01374R1

Dear Dr. Neff,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Robert Hoehndorf, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

4 May 2020

PONE-D-20-01374R1

Production location of the gelling agent Phytagel has a significant impact on Arabidopsis thaliana seedling phenotypic analysis

Dear Dr. Neff:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Robert Hoehndorf

Academic Editor

PLOS ONE