A Seed Vigor Test Through a Biospeckle Laser: A Comparison of Local and Global Analyses

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

- 1. This manuscript clearly states the limitations of traditional seed vigor tests, yet it provides limited physiological justification for selecting specific seed regions, particularly the vascular area and embryonic axis, for the local analysis approach...

- 2. The experimental design and data collection procedures are clearly described, but there is ambiguity regarding sample selection and representativeness.
The number of seeds tested seems reasonable, but further discussion is needed on how random points for local analysis within the seed area were selected, and additional information seems to be needed to increase reproducibility. What do you think?

- 3. The results section effectively presents clear statistical distinctions between high and low vigor groups, particularly emphasizing the embryonic axis’s superiority in distinguishing seed vigor levels. However, the findings from the vascular region are presented with limited physiological interpretation, leaving uncertainty around why this region did not yield significant differences. The manuscript could benefit from exploring potential explanations or alternative hypotheses for this unexpected result. What physiological mechanisms might explain the lack of detectable differences in the vascular region compared to the embryonic axis, and would adjusting imbibition timing or other experimental parameters potentially yield different outcomes?

- 4. The interpretation could be improved by discussing how the inclusion of the entire seed and surrounding optical table area might dilute or influence the biospeckle signal and its relation to seed vigor. 
Is it possible that global analysis inherently reduces sensitivity by incorporating irrelevant signals from physiologically inactive regions?
How can future studies also mitigate or control this effect?

- 5. In the Discussion section, the authors summarized their findings adequately, but there appears to be limited critical evaluation of methodological limitations and practical application issues.
While this manuscript successfully demonstrates the potential speed and sample size reduction benefits of BSL, it does not address broader practical issues such as consistency across different soybean varieties, standardization of procedures, and adaptability to different environmental conditions.
How might environmental conditions, genetic differences between seed batches, or handling practices affect the reproducibility and applicability of the Biospeckle laser method in real-world agricultural settings? What do you think?

- 6. Finally, the conclusion succinctly summarizes the key outcomes but lacks forward-looking considerations that would guide future research.
Please suggest any limitations or other research directions that could not be fully addressed in this study.

Below are suggested changes to the abstract.

L16 : “long durations” -> “extended durations”

L17-18 : “with the challenge to set…” -> “though standardization… remains a challenge”

L18-19 "the BSL test was then conducted…” -> “was then applied…”

L27 “compromise the vigor test” -> “affect the reliability of the vigor test”

I strongly recommend that you improve the clarity and quality of your manuscript by summarizing the above contents.

Thank you.

Author Response

Reviewer 1

We would like to thank you for your peer review that helped us to improve our manuscript. Your comments were addressed point-by-point, and the changes in the manuscript are highlighted in RED.

- 1. This manuscript clearly states the limitations of traditional seed vigor tests, yet it provides limited physiological justification for selecting specific seed regions, particularly the vascular area and embryonic axis, for the local analysis approach...

 Answer: We have increased, in the “Discussion” section, the information about the seed regions adopted in the work. Lines 421 – 424.

“The vascular area and the embryonic axis are regions of relevance in the germination process and that was the reason for choosing them during the BSL analysis. However, the two regions have distinct roles during seeds germination, and their different response after 24 hours of imbibition can be explained by the distinct activity of them in time.”

- 2. The experimental design and data collection procedures are clearly described, but there is ambiguity regarding sample selection and representativeness.
The number of seeds tested seems reasonable, but further discussion is needed on how random points for local analysis within the seed area were selected, and additional information seems to be needed to increase reproducibility. What do you think?

Answer: We have included a text in “Material and Methods” to explain the Gaussian distribution of the points and their representation of the region of observation. Lines 207 – 213.

“The selection of the points in a Gaussian distribution, that are responsible for build the THSP, allows the observation of the sample in a limited spot area. The random choice of the points provides the ability to cover the ROI, and the definition of their number associated with the standard deviation of the Gaussian function are relevant to assure a homogeneous covering of the ROI, but with great weight in its centre defined by the user. In addition, the replications are relevant to enhance the observation of the ROI.”

- 3. The results section effectively presents clear statistical distinctions between high and low vigor groups, particularly emphasizing the embryonic axis’s superiority in distinguishing seed vigor levels. However, the findings from the vascular region are presented with limited physiological interpretation, leaving uncertainty around why this region did not yield significant differences. The manuscript could benefit from exploring potential explanations or alternative hypotheses for this unexpected result. What physiological mechanisms might explain the lack of detectable differences in the vascular region compared to the embryonic axis, and would adjusting imbibition timing or other experimental parameters potentially yield different outcomes?

Answer: We agree with your point of view, and we have added additional comments about the vascular region in the “Discussion” section. It is in item 1.

- 4. The interpretation could be improved by discussing how the inclusion of the entire seed and surrounding optical table area might dilute or influence the biospeckle signal and its relation to seed vigor. 
Is it possible that global analysis inherently reduces sensitivity by incorporating irrelevant signals from physiologically inactive regions?

Answer: We have improved the discussion about the global analysis as suggested in the “Discussion” section. Lines 410 – 416.

“The application of BSL to entire speckle images—including areas less involved in germination, such as regions within the seed but distant from the embryonic axis, or even areas outside the seed boundaries—likely explains the lower significance of the Global analysis results compared to those of the Local analysis alone, like the embryonic axis. This suggests that the key issue is the reduced sensitivity of the Global analysis, caused by the influence of irrelevant regions that do not contribute meaningfully to vigor assessment.”

How can future studies also mitigate or control this effect?

 Answer: We have already stated about that in the “Discussion” section.

“The flexibility presented by the local analysis can, therefore, address the BA of heterogeneous elements such as seeds. The global analysis proposed by the use of the FTHSP [19] or by the autocorrelation [22] are relevant methodologies however when there is a homogeneous sample under analysis.”

- 5. In the Discussion section, the authors summarized their findings adequately, but there appears to be limited critical evaluation of methodological limitations and practical application issues.
While this manuscript successfully demonstrates the potential speed and sample size reduction benefits of BSL, it does not address broader practical issues such as consistency across different soybean varieties, standardization of procedures, and adaptability to different environmental conditions.
How might environmental conditions, genetic differences between seed batches, or handling practices affect the reproducibility and applicability of the Biospeckle laser method in real-world agricultural settings? What do you think?

Answer: We agree with your suggestions, and we inserted a paragraph in the end of the “discussion” section. Lines 445 – 452.

“This work represents an effort toward standardization of the seed vigor test using the BSL analysis, but in this case limited to the common bean seed (Phaseolus vulgaris L.). The protocol presented a clear design of the setup, the preparation of the seeds, according to the international rules [3], and the analysis in a laboratory of seed quality test without the influence of external environmental factors (e.g. sun light, wind, dust and mechanical vibration). Future research work should address the protocol adjustment or validation to different species and varieties. The time of imbibition and its relation to different regions within the seed must be considered in the standards for each species and varieties.”

- 6. Finally, the conclusion succinctly summarizes the key outcomes but lacks forward-looking considerations that would guide future research.
Please suggest any limitations or other research directions that could not be fully addressed in this study.

Answer: We introduced a paragraph in the end of the “Conclusion” section to address this point (Lines 479 – 482). Please, see that it was also presented in the end of the “Discussion” section. Lines 453 – 456.

“Future work should enlarge the number of species and varieties tested using the proposed protocol to create a solid standard for seed vigor test. It must be considered that each species and varieties can demand different time of imbibition to deliver the Local analysis.”

Below are suggested changes to the abstract.

L16 : “long durations” -> “extended durations”

L17-18 : “with the challenge to set…” -> “though standardization… remains a challenge”

L18-19 "the BSL test was then conducted…” -> “was then applied…”

L27 “compromise the vigor test” -> “affect the reliability of the vigor test”

Answer: We provided all the changes suggested.

I strongly recommend that you improve the clarity and quality of your manuscript by summarizing the above contents.

Answer: We thank you for the comments that we were pleased to accept about improving our work.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Overall, the authors' work provides strong experimental evidence for the application of Biospeckle Laser (BSL) technology in seed vigor assessment. By comparing global analysis and local analysis (especially the embryonic axis), the study effectively demonstrates the high applicability of BSL technology in seed vigor testing. However, despite laying the foundation for the application of BSL technology, there are still some issues worth further resolution and discussion.

1.It is suggested to emphasize the research contribution and potential future applications at the end of the abstract.

2.The conclusion of the introduction can concisely and clearly state the contributions or innovations of this study.

3.The paper does not provide specific values for light intensity and contrast, lacking quantitative descriptions of these parameters.

4.How can light intensity and contrast be ensured under different laboratory and equipment conditions? Is there any potential bias in seed vigor evaluation results due to varying light or contrast conditions?

5.Although the authors state that local analysis (especially the embryonic axis) is more effective than global analysis in distinguishing high and low vigor seeds, the reasons why global analysis is less effective than local analysis have not been thoroughly explained.

6.The authors mention that BSL technology is not yet fully standardized, but they do not discuss in detail how this issue may affect the research results. If the standardization issue is not addressed, it may affect the universal applicability and reliability of BSL technology in practical use.

7.The paper notes that improper region selection during local analysis may affect the test results. It is recommended that the authors further discuss in the discussion section how to adopt more precise region selection methods or propose measures to avoid selection bias, in order to enhance the accuracy of the analysis and ensure the reliability of the results.

8.If BSL technology is applied without knowing the seed vitality, how can the output results be used to determine whether the seeds are low or high in vitality? How is the vitality threshold set?

Author Response

Reviewer 2

We would like to thank you for your peer review that helped us to improve our manuscript. Your comments were addressed point-by-point, and the changes in the manuscript are highlighted in RED.

Overall, the authors' work provides strong experimental evidence for the application of Biospeckle Laser (BSL) technology in seed vigor assessment. By comparing global analysis and local analysis (especially the embryonic axis), the study effectively demonstrates the high applicability of BSL technology in seed vigor testing. However, despite laying the foundation for the application of BSL technology, there are still some issues worth further resolution and discussion.

1.It is suggested to emphasize the research contribution and potential future applications at the end of the abstract.

Answer: We have included in the “Abstract” a final sentence to address your suggestion, however limited by the 200 words.

“It was a relevant step toward standardization demanding additional tests in other species and varieties.”

2.The conclusion of the introduction can concisely and clearly state the contributions or innovations of this study.

 Answer: We included an additional paragraph in the end of the “Introduction” section.

“Therefore, this study proposes the adoption of the BSL local analysis as a standard procedure for seed vigor testing in Phaseolus vulgaris L., detailing the experimental setup, seed preparation, and image analysis methodologies. Additionally, recommendations for future research are provided to expand the standardization process to other seed species and varieties.”

3.The paper does not provide specific values for light intensity and contrast, lacking quantitative descriptions of these parameters.

 Answer: We have included a text with the values suggested for light intensity and contrast. Lines 306 – 310 and line 312.

“The adjusted thresholds for under-exposition of light were set in 35, on a gray scale (0 to 255), and for the over illumination was set in 220, on a gray scale. Thus, the blue circles represent the level of light equal or under 35, the red circles represent the level of light over 220 and the green circles represent the level of illumination between 35 to 220.”

“(…) high contrast expressed by the light grey squares, expressed from 100 to 255 on grey scale values.”

4.How can light intensity and contrast be ensured under different laboratory and equipment conditions? Is there any potential bias in seed vigor evaluation results due to varying light or contrast conditions?

Answer: The light intensity and contrast are relevant for the correct analysis of BSL, and the main issues are related to the under exposure and saturation (over exposure) of light on the sample surface or related to a blurred image of the speckle pattern, related to the low contrast. Therefore, the thresholds presented in this work, under the quality test, aimed to allow repeatability in different laboratories and using different equipment. We have included a paragraph regarding this issue. Lines 312 – 314.

“Therefore, once the level of illumination and the contrast of the speckle grains were within the boundaries, we can guarantee that the BSL indices were not compromised.”

5.Although the authors state that local analysis (especially the embryonic axis) is more effective than global analysis in distinguishing high and low vigor seeds, the reasons why global analysis is less effective than local analysis have not been thoroughly explained.

 Answer: We have included a text in the “Discussion” section to let it clear. Lines 410 – 416.

“The application of BSL to entire speckle images—including areas less involved in germination, such as regions within the seed but distant from the embryonic axis, or even areas outside the seed boundaries—likely explains the lower significance of the Global analysis results compared to those of the Local analysis alone, like the embryonic axis. This suggests that the key issue is the reduced sensitivity of the Global analysis, caused by the influence of irrelevant regions that do not contribute meaningfully to vigor assessment.”

6.The authors mention that BSL technology is not yet fully standardized, but they do not discuss in detail how this issue may affect the research results. If the standardization issue is not addressed, it may affect the universal applicability and reliability of BSL technology in practical use.

 Answer: We have included a paragraph in the “Discussion” section to address the standardization issue. Lines 445 – 452.

“This work represents an effort toward standardization of the seed vigor test using the BSL analysis, but in this case limited to the common bean seed (Phaseolus vulgaris L.). The protocol presented a clear design of the setup, the preparation of the seeds according to the international rules [3] and carrying out the analysis in a laboratory of seed quality analysis without the influence of external environmental factors (e.g. sun light, wind, dust and mechanical vibration). Future research work should address the protocol adjustment or validation to different varieties, and seeds form different species. The time of imbibition and its relation to different regions within the seed must be considered in the standards for each species and varieties.”

7.The paper notes that improper region selection during local analysis may affect the test results. It is recommended that the authors further discuss in the discussion section how to adopt more precise region selection methods or propose measures to avoid selection bias, in order to enhance the accuracy of the analysis and ensure the reliability of the results.

 Answer: We understand the point, and we have introduced a text in the “Discussion” section to deal with that. Lines 453 – 456.

“The analysis of vigor using the BSL must consider that the seeds of different species and varieties can vary the optimum point of observation regarding the germination process. Thus, further research shall consider the establishment adjustments in the proposed standard to each case.”

8.If BSL technology is applied without knowing the seed vitality, how can the output results be used to determine whether the seeds are low or high in vitality? How is the vitality threshold set?

 Answer: We introduced a text in the “Discussion” section to address properly this point. Lines 457 – 460.

“In addition, the proposed BSL analysis of seeds’ vigor must always calibrate the system using low- and high-vigor seeds in a preliminary step before the main analysis. And the calibration must be repeated always when a seed of a different species or variety is prepared to be tested.”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The quality and clarity of the manuscript have been improved compared to the previous version.

Thank you.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The author has made the necessary revisions and the paper is acceptable for publication.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The introduction notes that the proposed method has been reliably applied to various grains and beans; however, it does not establish a compelling rationale for conducting a localized analysis specific to the study's context. Furthermore, while the embryonic axis appears to be more responsive or “liable” in the results, the explanation for this observation lacks sufficient detail and scientific reasoning. To strengthen the study, it is recommended that the authors provide a clearer justification for local validation and offer a more thorough interpretation of the embryonic axis findings. Additionally, the study would benefit from exploring the comparative reliability of the Biochemical Stress Level (BSL) method against the standard Emergence Test, to validate its practical utility.

Line 102: standard procedure followed should be stated or referenced

Line 131-133: What protocol or reference was used?

Figure 2 & Figure 6: What the axis numbers, and index number of the color scheme represent

Figure 5: Where is ( c), line 278

Line 347-349: An explanation of why the technique was able to provide these results. What about the procedure that contributed?

Line 357: repetition

Reviewer 2 Report

Comments and Suggestions for Authors

The authors proposed a method to test the seed vigor through biospeckle laser. Taking the  Phaseolus vulgaris L. as case to study. But there are many limitations:

1.the purpose why you conduct this study, it's not clear.

2.Introduction part, the authors introduced the biospeckle laser technology, and there are studies in the testing of seeds, what's your difference among ref.8 and 10?

3.In addition, the authors introduced the methods like hyperspectral and X-ray methods, as i can know, these two methods are non-dectructive to seeds, they have more potential in the testing of seed vigour?

4.The methods and results are hard to read, maybe more figure introduction can help to solve this.

5.This study is destructive to seeds, no actual meaning for farmers

6.The big problem: Variety limited, only one variety are used, it can not represent other situation.

Comments on the Quality of English Language

It's hard for me to read this, such as some long sentence.

Reviewer 3 Report

Comments and Suggestions for Authors

It is important to evaluate the vitality of seeds quickly and objectively. This manuscript introduces the adoption of Biospeckle Laser (BSL) technique to shorten the time for detecting seed vigor, the experimental results indicated that this technique can be used as a rapid and resource-efficient method for assessing seed vigor. I believe this research is of scientific value and worthy of publication, the manuscript can be improved in the following points:

1. The 2nd paragraph introduced other detection technical means. However, the introduced content of different techniques was too little. The detection principles, advantages and disadvantages of different techniques should all be summarized. Hardly any explanation for the principle of BSL technique in detecting seed vigor can be found in the 3rd and 4th paragraphs either. In other words, it is necessary to explain clearly to the readers why this technique can detect seed vigor.
2. The division of each paragraph in the introduction needs to be adjusted. Currently, the logic is not strong enough.
3. The use of words ‘Local or Global Analysis’ in the title is inappropriate and can easily cause readers to be confused and misunderstand. Please use more appropriate expressions and words.
4. Please use the full name when using BOD for the first time.
5. In Line 122, the "En" was inconsistent with that in the formula.
6. Please check the case of the letter "K" in Equation 2, the last " I(K+1) " should be " I(k+1)".
7. What are the black and red circles in Figure 2?
8. In the study, the significance test was used to determine the seed samples with high vigor and low vigor. Why not train the classification models for discrimination by using image recognition and detection? Other techniques such as hyperspectral imaging can directly establish detection models to identify seed attributes. How will BSL technique be applied in the future?
9. In Table 1 and Table 2, please use integers to represent sown seeds and e merged seeds. It is recommended to use different shades of color to represent the quantity when filling the color blocks.
10. 10. The Figure 4 lacks a legend.

Reviewer 4 Report

Comments and Suggestions for Authors

The research work "Seeds Vigor Test Through Biospeckle Laser: A Case Study of Phaseolus vulgaris L. by Local or Global Analysis" evaluated the efficiency of local and global BSL analysis in bean seeds (Phaseolus vulgaris L.). The study proved the efficiency of BSL in testing the bean seeds' vigor. I do not see innovation in this study for the following reasons:

1- Based on the abstract, this research does not present any novel ideas, as BSL has already been utilized to assess seed vigor and detect infected seeds in several of the cited references, as well as in other studies not mentioned in the paper.

2- the methods used in the paper such as AVD1, AVD2, AVD3 have been extensively used in the literature.

3- Many other methods use BSL that should be tested and compared with the authors' method such as:

Evaluation of seed vigor through BSL using IM method, Material Engineering, and Mechanical Engineering. May 2016, 1159-1165.

Laser BSL technique for characterizing the impact of temperature and initial moisture content on seed germination, Optics and Lasers in Engineering,
Volume 153, 2022

4- No need to list the ESI  values of the emerging high and low-vigor seeds in Tables 1 and 2 if they were displayed using a box plot.

5- Why do the authors show only the analysis of variance (ANOVA) for AVD1? Although the authors indicated that AVD2, AVD3, and AVD4 are comparable to AVD1, they have to show them with a graph. 

6- What are Degrees of Freedom, Sum of Squares, and Mean of Squares? Explain them in the methods.

7- Why there is a remark under tables 4 and 6 not related to the content of the tables?