Effective Treatments for the Successful Establishment of Milkweed (Calotropis procera L.) under Water Deficit

Round 1

Reviewer 1 Report

Dear Authors,

The manuscript titled "Sustainable strategies for the cultivation of Calotropis procera L. under conditions of water deficit" by Mojtaba Dolatkordestani, Mansour Taghvaei, and Andrea Mastinu explores the interaction between seed coating and super absorbents in the cultivation of Calotropis procera L. (milkweed) under water deficit conditions. The study involves a greenhouse experiment with various treatment factors, including coating materials, growth media, and field capacity regimes. Here is a review of the manuscript:

Strengths:
Relevance of Research: The study addresses an important and relevant topic in agricultural research - finding sustainable strategies to cultivate plants under conditions of water deficit, which is particularly crucial in arid regions.

Experimental Design: The factorial arrangement of treatments, including different coating materials, growth media, and field capacity regimes, adds depth to the study's design. This approach allows for a comprehensive examination of how these factors interact to influence plant growth.

Data Collection: The authors have collected a wide range of data, including morphological and physiological parameters, enzyme activities, and chlorophyll content. This comprehensive data collection strengthens the study's ability to draw meaningful conclusions.

Hypothesis and Research Objective: The manuscript clearly states the hypothesis and research objectives, providing a clear framework for the study. The hypothesis regarding the use of super absorbent materials and organic fillers to mitigate water stress during germination is well-founded and compelling.

Areas for Improvement:

Abstract: please highlight the practical significance of the results obtained.

Material and Methods: laboratory assessment of seed germination parameters (mainly germination capacity) performed under optimal conditions could further enrich the results and expand the possibility of their interpretation.

Results: Please ensure that all tables and figures are self-explanatory and do not require readers to search for abbreviations in the manuscript's text.

1) Table 2 - please explain EC.

2) Tables 3 and 5 - please explain FC and what the value after +/- means.

3) Line 235 - S4 is missing.

4) Table 4 - please explain Chl and FC and what the value after +/- means.

Discussion: The discussion of results should be more detailed and focused on explaining the implications of the findings. Specifically, the manuscript could provide more insight into the mechanisms by which super absorbent polymers and seed coatings affect plant responses to water deficit.

Conclusions: The conclusions section should be more explicit about the practical implications of the findings for sustainable cultivation of Calotropis procera L. under water deficit conditions. How can these results be applied by farmers or researchers?

Language and Clarity:
The manuscript is generally well-written, but there are instances of awkward phrasing and language that could be further refined for clarity and readability. Proofreading for grammatical and typographical errors is recommended.

Ensure that the manuscript is appropriately formatted according to temptate.

In summary, the manuscript addresses an important research question and presents valuable data on the cultivation of Calotropis procera L. under water deficit conditions. With some improvements in presentation, discussion, and clarity, it has the potential to contribute significantly to the field of sustainable agriculture and water management in arid regions. Therefore, I believe that the editors of Land should consider publishing this manuscript.

Author Response

REVIEWER#1

Dear Authors,

The manuscript titled "Sustainable strategies for the cultivation of Calotropis procera L. under conditions of water deficit" by Mojtaba Dolatkordestani, Mansour Taghvaei, and Andrea Mastinu explores the interaction between seed coating and super absorbents in the cultivation of Calotropis procera L. (milkweed) under water deficit conditions. The study involves a greenhouse experiment with various treatment factors, including coating materials, growth media, and field capacity regimes. Here is a review of the manuscript:

Strengths:
Relevance of Research: The study addresses an important and relevant topic in agricultural research - finding sustainable strategies to cultivate plants under conditions of water deficit, which is particularly crucial in arid regions.

Experimental Design: The factorial arrangement of treatments, including different coating materials, growth media, and field capacity regimes, adds depth to the study's design. This approach allows for a comprehensive examination of how these factors interact to influence plant growth.

Data Collection: The authors have collected a wide range of data, including morphological and physiological parameters, enzyme activities, and chlorophyll content. This comprehensive data collection strengthens the study's ability to draw meaningful conclusions.

Hypothesis and Research Objective: The manuscript clearly states the hypothesis and research objectives, providing a clear framework for the study. The hypothesis regarding the use of super absorbent materials and organic fillers to mitigate water stress during germination is well-founded and compelling.

Areas for Improvement:

Abstract: please highlight the practical significance of the results obtained.

RE: For quantified and highlight the results two following sentence rewrite added to abstract

The highest chlorophyll (0.68), leaf area (15.41), shoot dry weight (31.47, and root dry weight (19.87) were observed when 4 g of super absorbent was used in the growth medium sever water deficit (25% FC).

The vermicompost coating with the use of a super absorbent polymer (4 g) per kg soil was the best to establish milkweed under mild (50% FC) and severe water deficits (25% FC).

Material and Methods: laboratory assessment of seed germination parameters (mainly germination capacity) performed under optimal conditions could further enrich the results and expand the possibility of their interpretation.

RE: Fortunately, the reaction of the germination of Calotropis procera L to drought stress has already been investigated and article’s has been published.

Taghvaei. M, Sadeghi. H, Bazrafcan M. 2017. The effect of drought stress on the seed germination of Calotropis procera L. and improvement of drought tolerance. Journal and plant researches (Iranian journal of biology). Vol. 30 No. (1).

Results: Please ensure that all tables and figures are self-explanatory and do not require readers to search for abbreviations in the manuscript's text.

RE: Thanks to the note of the honorable referee, the entire text was re-examined

• Table 2 - please explain EC.

RE: It was changed as follows

Electrical conductivity (EC)

• Tables 3 and 5 - please explain FC and what the value after +/- means.

RE: It was added as follows in the title and subtitle of the table respectively

S 1 = Soil, S2 = Sand + Soil 1:1 ratio, S3= 2 g super absorbent per kg soil, S4=4 g super absorbent per kg soil (S3),and FC= field capacity.

For each variable, mean (± standard deviation) values followed by the same letters (small letters for interactions and capital letters for means) were not significantly different using LSD at the 5% level.

• Line 235 - S4 is missing.

RE:

The titles of all tables were corrected

S 1 = Soil, S2 = Sand + Soil 1:1 ratio, S3= 2 g super absorbent per kg soil, S4=4 g super absorbent per kg soil (S3), and FC= field capacity.

• Table 4 - please explain Chl and FC and what the value after +/- means.

RE:in titles of table 4 were corrected as follows

S1 = Soil, S2 = Sand + Soil 1:1 ratio, S3= 2 g super absorbent per kg soil, S4=4 g super absorbent per kg soil (S3), chl= chlorophyll and FC= field capacity.

Discussion: The discussion of results should be more detailed and focused on explaining the implications of the findings. Specifically, the manuscript could provide more insight into the mechanisms by which super absorbent polymers and seed coatings affect plant responses to water deficit.

RE:

To further justify the role of super absorbent, the following sentence was added to the results and discussion section

It has been reported that superabsorbents can absorb water several times their own weight and provide it to plants under drought stress [43]. The consequences of soil moisture variability during plant establishment could be reduced by increasing soil water holding capacity (Patra et al., 2019 ). The use of hydrogel "superabsorbent polymers" (SAPs) to increase topsoil water holding capacity (WHC) is an appropriate management tool. SAPs absorb a significant amount of water and improve the topsoil's ability to store water.

Conclusions: The conclusions section should be more explicit about the practical implications of the findings for sustainable cultivation of Calotropis procera L. under water deficit conditions. How can these results be applied by farmers or researchers?

RE: The following sentence was added to the end of the discussion for more clarity
Therefore, to increase the percentage of success in the establishment of this plant, instead of directly planting the seeds, the farmer can put the seeds of this plant inside the seed balls made of vermicompost and super absorbent and then plant them in the soil.

Language and Clarity:
The manuscript is generally well-written, but there are instances of awkward phrasing and language that could be further refined for clarity and readability. Proofreading for grammatical and typographical errors is recommended.

RE: Thanks to the accuracy of the reviewer's opinion, the article was again checked for spelling and grammar for more accuracy

Ensure that the manuscript is appropriately formatted according to temptate.

RE: Compliance with the journal’s format was checked

In summary, the manuscript addresses an important research question and presents valuable data on the cultivation of Calotropis procera L. under water deficit conditions. With some improvements in presentation, discussion, and clarity, it has the potential to contribute significantly to the field of sustainable agriculture and water management in arid regions. Therefore, I believe that the editors of Land should consider publishing this manuscript.

Reviewer 2 Report

The manuscript entitled “Sustainable strategies for the cultivation of Calotropis procera L. under conditions of water deficit” is interesting but has many flaw information that need to be discussed. Unfortunately for me has no sufficiently scientifically sound results to accept it as it is with this experimental design. 

Introduction - Since Calotropis procera is a perennial plant, a shrub, it is a little bit strange the authors used examples of crop to justify their work approach. There are different works on using absorbent products that could be much more appropriate. This kind of methodologies are used in different restoration programs. None are referred. Work hypothesis is not understandable. Please rewrite and clarify it.

Methodology - You need to be much more detailed.

You said “After preparing the 100 halves ball, one seed was placed between two halves balls (Figure 1), and the prepared balls were transferred to 5 L volume pots filled with 4 kg of soil” If those halves ball are dried how could you assured that when you insert the balls in the pots they kept the seeds inside? Also, it would be important to say how many seeds have you inserted in each pot.

The chemical properties of composts as well as soil should be presented in mg/kg and not some as % others as mg/kg. Only organic matter is ok in %. 

The soil you inserted has a relatively high organic matter. From where did you get this soil? Is it in native ecosystems or was this prepared?

Using so many soil combinations field capacity should be very different of each other - clarify. Data comparisons sounds also very tricky, since during the experiment plants germination are facing very different soil nutrients availability, not only water.

How many pots did you prepare? How many plants did you get at the end?

Results - why you didn’t give data of germination? You started saying that this plant is very sensitive in germination phase of their life cycle. How many survived ? Data you presented are the result of how many samples ? For restoration purposes more than plant physiological responses should be, germination, growth rates, total biomass.

How can you assure that vermicompost is the best due to its capacity of holding water? This can be only a part of interpretation since this is the most rich in organic matter (a natural holding water) and nutrients. No proper comparisons can be made.

Author Response

The manuscript entitled “Sustainable strategies for the cultivation of Calotropis procera L. under conditions of water deficit” is interesting but has many flaw information that need to be discussed. Unfortunately for me has no sufficiently scientifically sound results to accept it as it is with this experimental design. 

Introduction - Since Calotropis procera is a perennial plant, a shrub, it is a little bit strange the authors used examples of crop to justify their work approach. There are different works on using absorbent products that could be much more appropriate. This kind of methodologies are used in different restoration programs. None are referred. Work hypothesis is not understandable. Please rewrite and clarify it.

RE: Thanks to the referee's attention to the perenniality of this plant, in the process of ecosystem restoration the emphasis is on the establishment of perennial and permanent plants, because after the establishment they remain in the ecosystem for a long time and play the role of a nurse plant, creating a safe place for the entry of other species. They facilitate the growth of plants and during the ecological succession the following species are gradually established and form new vegetation, therefore we emphasize the establishment of this plant.

Therefore, at the request of the respected referee, we have rewritten the hypothesis as below, which I hope will be convincing

Our hypothesis is since it seemed that the reason for the decrease in green percentage in the pasture is the type of growth medium or soil, that percentage of silt is high and the percentage of organic matter is very low, which cannot absorb a significant amount of water from accidental spring rains and the use of super absorbent and organic matter can increase the absorption of spring rain. That the supe-absorbent material can absorb a significant amount of rainwater and provide it to the plant for a longer period, thus reducing the water stress during the emergence stage.

Methodology - You need to be much more detailed.

You said “After preparing the 100 halves ball, one seed was placed between two halves balls (Figure 1), and the prepared balls were transferred to 5 L volume pots filled with 4 kg of soil” If those halves ball are dried how could you assured that when you insert the balls in the pots they kept the seeds inside? Also, it would be important to say how many seeds have you inserted in each pot.

RE:

For more clarity, I need to explain that these coatings are made in the lab and then dried. In the spring, almost before the spring rains (planting time), instead of planting the seeds directly in the soil, the seeds are placed inside the cover and placed in the planting hole, and they will be covered with a thin layer of soil until after the spring rains. And the absorption of rainwater, the seed starts to grow and emerge slowly from the middle of the cover, so for greater clarity, I have added an abstract graphic to the materials and method section. For more clarity, I need to explain that these coatings are made in the lab and then dried. In the spring, almost before the spring rains (planting time), instead of planting the seeds directly in the soil, the seeds are placed inside the cover and placed in the planting hole, and they will be covered with a thin layer of soil until after the spring rains. With the absorption of rainwater, the seed starts to grow and emerge slowly from the middle of the cover, so for greater clarity, I have added a graphical abstract to the materials and methods section. If more clarification is needed, let me know. Since we have performed this test in the greenhouse, we have used 5 kg pots instead of soil.

So, this sentence was rewritten as below if need more explanation please let me know

Then, 2 g of each dough coating material was injected into a plastic half-circle (with a diameter of 5 cm), and the molds were dried at room temperature. They were kept in a sterile environment until the beginning of the experiment(Figure 1). It should be noted that in the middle of one of the molds, there was a 4 mm deep hole for placing seeds. At the time of planting one seed was placed between two halves of molds and was planted in 5 L volume pots filled with growth medium'' based on experimental treatments'' (with one hole drilled in the bottom of each pot for drainage)(Figure 1), and after emergence, one of them was removed. To apply water stress, the field capacity of the soil was determined.

The chemical properties of composts as well as soil should be presented in mg/kg and not some as % others as mg/kg. Only organic matter is ok in %. 

RE: The above items were modified in Table 2

The soil you inserted has a relatively high organic matter. From where did you get this soil? Is it in native ecosystems or was this prepared?

RE: The percentage of organic matter in Table 1 is a typographical error and was corrected. If you notice, we have mentioned in the introduction that there is very little organic matter in these areas.

And we have taken this soil from the studied area so that the results we get are more practical

In addition to the fact that we mentioned this issue in the hypothesis, I also added the following sentence in the introduction to emphasize this issue

Dryland soils are characterized by low organic matter content, low waterholding capacity, low infiltration, and low fertility, which reduces the percentage of seedling establishment due to low water maintenance.One way to improve seedling emergence and early seedling development is to use seed coating fillers [Amirkhani, etal] to increase the water stored in the soil for a longer period of time [Beigi, etal]. Organic materials such as peat moss and vermicompost can be used as soil water stores to increase moisture around the seed.

Using so many soil combinations field capacity should be very different of each other - clarify. Data comparisons sounds also very tricky, since during the experiment plants germination are facing very different soil nutrients availability, not only water.

How many pots did you prepare? How many plants did you get at the end?

RE: regarding the comparisons, I must mention that we had a control treatment and we measured and compared the traits studied in the control treatment and other treatments in order to know the effect of the difference in the culture medium and seed cover, and the superabsorbent

About the field capacity: The field capacity was measured for the treatments

I added this item to the materials and methods to clarify the issue

It should be noted that in the middle of one of the molds, there was a 4 mm deep hole for placing seeds. At the time of planting one seed was placed between two halves of molds and was planted in 5 L volume pots filled with growth medium'' based on experimental treatments'' (with one hole drilled in the bottom of each pot for drainage)(Figure 1).To apply water stress, the field capacity of the soil was determined.

In this experiment, we have examined plant growth indicators in pots and water treatments were applied 15 days after emergence, for greater clarity, we added the following sentence to the materials and method.

After 15 days ofsowing, water deficit treatments) 25, 50, and 100% of the field capacity( were carried out for three months and then the plantswere harvested to determine indices. Every alternate day, to apply the filed capacity the pots were weighed and water treatments were applied to the weight base and water was added to achieve the target soil moisture

The number of pots was 192 (4*4*3*4) C, M, I, and replication, 2 seeds were planted in each pot and after emergence, one of them was removed.

To express the number of plants in the pot, the following sentence was added to the materials and method section

At the time of planting two seed was placed between two halves of molds and was planted in 5 L volume pots filled with growth medium'' based on experimental treatments'' (with one hole drilled in the bottom of each pot for drainage)(Figure 1),and after emergence, one of them was removed.To apply water stress, the field capacity of the soil was determined.

Results - why you didn’t give data of germination? You started saying that this plant is very sensitive in germination phase of their life cycle. How many survived ? Data you presented are the result of how many samples ? For restoration purposes more than plant physiological responses should be, germination, growth rates, total biomass.

 RE: As the respected reviewer also noted, in the greenhouse studies where the seeds are grown in pots, we have the trait of emergence, which we refused to present in order to avoid the length of the article, and it was added to your request.

However, at your request, three other important traits such as emergence percentage, leaf-specific weight, and root-to-shoot ratio were added to the article.

How can you assure that vermicompost is the best due to its capacity of holding water? This can be only a part of interpretation since this is the most rich in organic matter (a natural holding water) and nutrients. No proper comparisons can be made.

RE: We understand what you mean, but in this test these materials were evaluated in terms of saturated water absorption and water retention time (see the summary graph we added to the Materials and Methods section) and compared to the control.

Reviewer 3 Report

Tables should be improved to highlight results.

Author Response

Tables should be improved to highlight results.

RE: Changes have been made to the tables and three more attributes have been added based on the judges' opinions, and Table 2 has been modified and the Analysis of Variance table has been added.

Round 2

Reviewer 2 Report

At the end you answered better to my questions in your responses than in the paper. Why did you omitted the total number of plants you dealt with? 

Author Response

Reviewer#2

At the end you answered better to my questions in your responses than in the paper. Why did you omitted the total number of plants you dealt with?

AUTHORS: We thank the Reviewer for his care in reviewing our manuscript. We apologize for omitting the total number of plants. We have now integrated the information into the text:

Fresh 500 capsules of Calotropis procera L. were collected in August 2022 from one of its natural habitats in the rangelands of Sarbaz