Hemp (Cannabis sativa L.) Tolerates Chelator Stress Showing Varietal Differences and Concentration Dependence
Round 1
Reviewer 1 Report
In this article, authors identified seed germination-related traits in seven genotypes of hemp under various concentrations of chelators. Based on this experiment, the authors selected two genotypes to evaluate their physiological and biochemical performance under various concentrations of chelators. The provided results are not sufficient for publication. Some of the experiments are needed to improve the manuscript. I have raised more than 15 questions to improve the manuscript. So authors should try to do some experiments related to this study that I asked.
1. How did the authors select various concentrations of EDTA and CA for their experiment?
2. Why authors did not select 25 mmol·L-1 chelators on the growth and physiological parameters of two hemp cultivars. Authors should check the physiological and biochemical changes under 25 mmol·L-1 chelators in the two cultivars
3. Why did the other not analyze other biochemical traits such as CAT, DHAR, proline, sugar contents, and more? The authors try to include all the necessary biochemical traits.
4. What about the yield data? Yield data is more important for this study. Authors should include this.
5. Authors should analyze all the biochemical traits in root tissues. Furthermore, try to collect root architectural modification
6. Without checking heavy metal contents in root and shoot tissues how did the authors confirm CA (2 mmol·L-1 + cultivar BM might be a promising combination for reconciling plant growth and heavy metal remediation efficiency. So, authors should check heavy metal concentrations (Cd, Pb, Ni, Co and Zn) in different plant tissues.
7. Authors should italicize the botanical name of Hemp in the title
8. Authors can add the botanical name of hemp in L. No. 10
9. L. No. 64; authors should write a full form of Cd, Pb and Zn
10. Authors should mention the size of the pot and what materials (soil/vermicompost/any other) are used for plant growth in the materials and methods section.
11. L. No. 92; Did the authors not supply any micronutrients for plant growth?
12. L. No. 97; control (CK)- Is water or any other nutrients? What is CK?
13. Authors should add the full form of SS, df, MS, F, and others as a footnote in the table. Authors should correct these in all the tables
14. Authors should write all the figure legends in detail.
15. Chlorophyll content a or b or total chlorophyll? what is chlorophyll alone?
16. Authors should include all the raw data of physiological, biochemical and other traits as a supplemental file.
17. Authors should submit morphological changes of hemp cultivars as a main/supplemental figure under various concentrations of chelators. All the figures should be included
Nil
Author Response
- How did the authors select various concentrations of EDTA and CA for their experiment?
Re: Thank you very much for your comments. We conducted a preparatory test on hemp with reference to the usual chelator application concentrations. Based on the results of the preparatory test, together with considerations of the likelihood of execution of the test, the chelator concentration levels in the study were set. The information was added to the text in part 2.2.
- Why authors did not select 25 mmol·L-1 chelators on the growth and physiological parameters of two hemp cultivars. Authors should check the physiological and biochemical changes under 25 mmol·L-1 chelators in the two cultivars
Re: Based on the results of our preparatory experiments, the 25 mmol·L-1 chelating agent was too toxic to hemp growth and plants survival for the necessary tests to be conducted, so the 25 mmol·L-1 concentration was omitted from the formal experiments. The information was also added to the text in part 2.3.
- Why did the other not analyze other biochemical traits such as CAT, DHAR, proline, sugar contents, and more? The authors try to include all the necessary biochemical traits.
Re: Thank you very much for your suggestions. We have carefully referred to some relevant literatures in conducting this study, and different literature reported different biochemical traits, and based on the factors that can reflect the treatment effects and that these indicators are related to plant growth and stress tolerance, as well as the feasibility of implementation, we chose to test these indicators reported in the paper. At the same time, we sincerely believe that it is difficult for us to cover and test all possible indicators in one study. We appreciate your understanding.
- What about the yield data? Yield data is more important for this study. Authors should include this.
Re: Although we did not investigate economic yields of hemp (e.g., fiber yields), the effects of applying chelating agents on biomass are provided in our paper, and for focusing on phytoremediation of heavy metal contamination of soils, the biomass of the plants is more important than the economic yields (e.g., hemp fiber yields).
- Authors should analyze all the biochemical traits in root tissues. Furthermore, try to collect root architectural modification
Re: Thank you very much for your suggestions. We are very sorry that we have not been able to analyze all the biochemical traits in root tissues and collect root architectural modifications. This has to be a pity in the execution of our experiment, although the findings reported in our paper can also illustrate what needs to be illustrated.
- Without checking heavy metal contents in root and shoot tissues how did the authors confirm CA (2 mmol·L-1 + cultivar BM might be a promising combination for reconciling plant growth and heavy metal remediation efficiency. So, authors should check heavy metal concentrations (Cd, Pb, Ni, Co and Zn) in different plant tissues.
Re: Thank you very much for your comments. The status of Pb uptake in hemp after application of chelating agents is reported in our published paper (Hu et al., 2022)，although hemp showed the best remediation efficacy when 5 mmol·L−1 EDTA or 10 mmol·L−1 CA was added in Pb contaminated substrate, consideration of the facts that growth of hemp, such as plant height, stem diameter and biomass, increased when 2 mmol·L−1 EDTA and 2~5 mmol·L−1 CA was added respectively, and the growth indexes of hemp decreased in varying degrees under other chelator concentrations, as well as the possible unsafe nature of EDTA, combined with the results of this trial’s focus on the effect of chelating agents on the growth of industrial hemp (including the higher tolerance of cultivar BM to chelating agents), we concluded that CA (2 mmol·L-1 ) + cultivar BM might be a promising combination for reconciling plant growth and heavy metal remediation efficiency. Some of the explanations were added also in the paragraph concerned in part 4 (Discussion).
- Authors should italicize the botanical name of Hemp in the title
Re: We italicized the botanical name of hemp in the title following your suggestion.
- Authors can add the botanical name of hemp in L. No. 10
Re: Thanks for your suggestion. Since the botanical name of hemp was given in the title, it may be omitted in the text?
- L. No. 64; authors should write a full form of Cd, Pb and Zn
Re: We wrote the full name of Cd, Pb and Zn in the text.
- Authors should mention the size of the pot and what materials (soil/vermicompost/any other) are used for plant growth in the materials and methods section.
Re: The substrate used in the pot culture was expressed in part 2.1 L89-90, that was commercial peat produced by JIFFY Company, Norway. The size of pot used in the experiment was noted in part 2.3 L106-107 (now L108-109), that was plastic pot with tray of 40 cm diameter and 26 cm height.
- L. No. 92; Did the authors not supply any micronutrients for plant growth?
Re: We did not add micronutrients to the substrate because the commercial substrate used already had micronutrients in it, and because of the short growth duration (66 days) of the industrial hemp in this trial, there was no apparent micronutrient deficiency as seen in the normal growth of the control plants.
- L. No. 97; control (CK)- Is water or any other nutrients? What is CK?
Re: Here CK meant no chelator was used, i.e. the concentration of chelator is zero. An explanation was also added to the text.
- Authors should add the full form of SS, df, MS, F, and others as a footnote in the table. Authors should correct these in all the tables
Re: We added the full forms of SS (sum of squares), df (degrees of freedom), MS (mean square), F (F-value), and others as footnotes in the table; and correct all these in the tables.
- Authors should write all the figure legends in detail.
Re: We furtherly modified all of the figure legends and added some necessary information.
- Chlorophyll content a or b or total chlorophyll? what is chlorophyll alone?
Re: The SPAD reads determined by the instrument characterized the total chlorophyll content.
- Authors should include all the raw data of physiological, biochemical and other traits as a supplemental file.
Re: We prepared all the raw data of physiological, biochemical and other traits as a supplemental file.
- Authors should submit morphological changes of hemp cultivars as a main/supplemental figure under various concentrations of chelators. All the figures should be included
Re: We are grateful to your suggestions. Unfortunately, we did not collect a full set of morphological images, with only individual temporal photographs showing the status of the trial (we can provide them if needed). We have not completely understood “All the figures should be included”, we sincerely apologize for this.
Reviewer 2 Report
The authors carried out and presented the results of good experimental work, which will improve the efficiency of phytoremediation of soils contaminated with heavy metals.
At the same time, the following questions need to be answered:
1. Does the risk of heavy metal contamination of ground and surface water increase when citric acid is used as a chelator?
2. How do the chelators used in various concentrations affect the phytoextraction of heavy metals from the soil?
3. Can it be argued that the mechanisms of action of chelators on hemp will be similar for other types of extractor plants.
Comments for author File: 
Comments.pdf
Editing of the English language is required, many sentences are difficult to understand and somewhat "overloaded".
Author Response
- Does the risk of heavy metal contamination of ground and surface water increase when citric acid is used as a chelator?
Re: More experimental analysis is needed to confirm this problem, but in another experiment we found no significant heavy metal differences in soil before and after citric acid application, so the risk of increasing contamination in the soil with citric acid was limited.
- How do the chelators used in various concentrations affect the phytoextraction of heavy metals from the soil?
Re: Our published paper on this issue (Hu et al., 2022) showed the increase in the absorption of Pb in hemp plants after the use of chelating agents, although only Pb was measured in the test; which was also explained in L66-69 in this paper.
- Can it be argued that the mechanisms of action of chelators on hemp will be similar for other types of extractor plants.
Re: More experiments are needed to prove this problem, but studies on other plants have reported that the use of chelating agents can increase the absorption of heavy metals by specific plants (Jalali and Khanboluki, 2007; Meers et al. 2008).
Editing of the English language is required, many sentences are difficult to understand and somewhat "overloaded".
Re: We intensively edited the English language throughout the full text, hope it now would be satisfied.
Round 2
Reviewer 1 Report
The authors only addressed all the minor comments they did not address major comments. I suggested to the authors to do some experiments but they did not perform. So the current manuscript is not improved. So reconsider after major revision. If the authors are not ready to perform further experiments, editors can make further decisions for this manuscript.
NIl
Author Response
Dear expert,
We are grateful to your help in processing our manuscript, while upon to the "major revision" suggestions based on our resubmitted revised manuscript "…… I suggested to the authors to do some experiments but they did not perform. So the current manuscript is not improved. So reconsider after major revision……"
We carefully read and understood the suggestions that need us to do further experiments, such as (a) analyzing other biochemical traits such as CAT, DHAR, proline, sugar contents, and more; and (b) analyzing all the biochemical traits in root tissues, furthermore, try to collect root architectural modification.
We express our sincere thanks to your suggestions and here we honestly respond to them at this moment. We are grateful to your understanding.
(a) To finish the suggested experiments need at least half a year or more time, therefore we are not in a position to do this at the moment, although test of those traits would add data in quantity. In the fact, we chose to test the indicators reported in the paper manuscript, based on the factors that can reflect the treatment effects and that the indicators are related to plant growth and stress tolerance, as well as the feasibility of implementation. At the same time, we sincerely think that it is difficult for us to cover and test all possible indicators in one study.
(b) As for the biochemical traits in root tissues and root architectural modifications, there should be some changes in root tissues and root architectures in responding to the treatment of chelators of different concentrations, which was partially illustrated by the changes in radicle length of germinated hemp seeds under chelator treatment reported in our manuscript, but we are very sorry for being not able to analyze all the biochemical traits in root tissues and collect root architectural modifications. This has to be a pity in the execution of our experiment, although the findings reported in our paper can also illustrate what was needed to be illustrated—impacts of certain chelating agents on seed germination and plant growth of industrial hemp.
Yours
Fei-hu Liu, on behalf of the co-authors
