Review Reports - PEF Priming of Seeds for Microgreen Production: A Comparative Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript investigates the effects of PEF pre-treatment on seed germination, growth, and post-harvest quality of three microgreen species (beetroot, arugula, basil). The experimental design is complete, and the data are substantial. However, issues remain in the logical flow, data presentation, and focus of discussion, which require revision before consideration for acceptance. Specific comments are as follows:

The final paragraph of the Introduction mentions that many scholars have studied PEF effects on various crops. Should the key findings of these previous studies be briefly summarized? The research objectives of this study should be clearly articulated in the context of microgreen cultivation, and the contribution of this work to expanding the knowledge of PEF effects on crop growth should be further justified.
The selection of the three specific electric field strength levels (2, 3, 4 kV/cm) in the Materials and Methods section lacks sufficient justification. For instance, Ahmed et al. (2020) explicitly used a range of 2–6 kV/cm in their study on wheat plantlet juice, which showed significant increases in antioxidant compounds. The rationale for choosing the narrower 2–4 kV/cm range in this study is not convincingly explained.
The Materials and Methods section heavily cites literature from 30-40 years ago to support the methodologies. The appropriateness of this is questionable. For example (and this is just one instance among several), lines 163-172 justify the acceptance of the W-S index formula by citing very old sources. Is it necessary to reference the original sources to this extent? Consider supplementing with more recent references where applicable.
In the Results section (e.g., Section 3.1.1), the description of statistical results (e.g., F-values, p-values, ηp²) is overly verbose and repetitive, hindering the readability of the key findings. It is recommended to streamline the presentation of statistical details and focus more on interpreting the biological significance.
The manuscript lacks in-depth analysis of the experimental results. For instance, the observed species-specific effects of PEF on chlorophyll content—positive for basil but negative for beetroot and arugula—are not explained mechanistically. Furthermore, the findings are not sufficiently compared and contrasted with results from existing published literature, which undermines the credibility of the study's conclusions.
The caption for Figure 1 (Chlorophyll and Carotenoid content) states "Statistical comparisons were conducted within each colour group," which is unclear. It should explicitly state that comparisons were made between different treatment groups within each species.
The Sensory Analysis section (3.3) notes that differences between groups were not statistically significant, yet considerable text is devoted to describing minor numerical variations on the radar charts. Given the lack of statistical significance and likely minimal practical relevance of these differences, this section should be condensed, focusing on the primary conclusion of "no significant effect."

Author Response

Comments 1: Specific comments are as follows: The final paragraph of the Introduction mentions that many scholars have studied PEF effects on various crops. Should the key findings of these previous studies be briefly summarized? The research objectives of this study should be clearly articulated in the context of microgreen cultivation, and the contribution of this work to expanding the knowledge of PEF effects on crop growth should be further justified.

Response 1: We appreciate the Reviewer’s insightful suggestion. In the revised version, we have expanded the final paragraph of the Introduction to briefly summarize the main findings of previous studies on the effects of PEF on seed germination and plant development. Furthermore, the research objectives are now more clearly articulated within the context of microgreen cultivation. Lines 101-119 (highlighted in yellow)

Comment 2: The selection of the three specific electric field strength levels (2, 3, 4 kV/cm) in the Materials and Methods section lacks sufficient justification. For instance, Ahmed et al. (2020) explicitly used a range of 2–6 kV/cm in their study on wheat plantlet juice, which showed significant increases in antioxidant compounds. The rationale for choosing the narrower 2–4 kV/cm range in this study is not convincingly explained.

Response 2: The selection of the electric field strengths (2, 3, and 4 kV/cm) was guided by both biological and economic considerations. From a biological standpoint, previous studies have demonstrated that the effects of PEF are highly species-dependent and follow a threshold-type response. In fact, Ahmed et al. (2020) reported maximum beneficial effects at 6 kV/cm; however, the results of Leong et al. (2016) contradict those findings, showing a reduction in coleoptile length (−6 mm) and primary leaf growth (−10 mm), with 2 kV/cm. In addition, our preliminary case study (conference poster) with Phaseolus vulgaris L., in which seeds were subjected to 2 kV/cm and 7 μs monopolar pulses, showed an increase in germination rate from 40% to 64% three days after PEF + sowing. A narrower range was therefore considered more appropriate to guarantee sublethal electropriming conditions. Furthermore, other factors should be taken into consideration, such as the scalability of the equipment and the cost associated, namely relative to the increase of the voltage of the equipment. Increasing the electric field two-fold means that the Joule heating increases four times and the cost of the equipment is higher, as the electrical current increases two-fold also. Thus, the selected range of protocols were designed to balance biological efficiency and practical applicability for possible future adoption of PEF in microgreens. (Lines 153-157 were added for clarification)

Comment 3: The Materials and Methods section heavily cites literature from 30-40 years ago to support the methodologies. The appropriateness of this is questionable. For example (and this is just one instance among several), lines 163-172 justify the acceptance of the W-S index formula by citing very old sources. Is it necessary to reference the original sources to this extent? Consider supplementing with more recent references where applicable.

Response 3: The reviewer is correct in noting that some of the references cited are older. In our vision, those references (i.e. Reddy 1985 and Walker-Simmons, 1987) were retained because they first proposed and formulated the formula used, which is still used in the present. While we have mentioned two recent studies (Kader, 2005, Al-Ansari & Ksiksi (2016), as per the reviewer’s advice, and to ensure recent methodological relevance, recent studies and applications of the same formula have been included to strengthen its continued validity and use in modern seed and microgreen germination research. (alterations: lines 191-194; line 198 &199; line 209-2011, lines 226-229, line 260 & 272).

Comment 4: In the Results section (e.g., Section 3.1.1), the description of statistical results (e.g., F-values, p-values, ηp²) is overly verbose and repetitive, hindering the readability of the key findings. It is recommended to streamline the presentation of statistical details and focus more on interpreting the biological significance.

Response 4: We thank the reviewer for the comment. The Results section has been revised to remove redundant reporting of F-values, p-values, and ηp², improving readability (lines 353-388)

Comment 5: The manuscript lacks in-depth analysis of the experimental results. For instance, the observed species-specific effects of PEF on chlorophyll content—positive for basil but negative for beetroot and arugula—are not explained mechanistically. Furthermore, the findings are not sufficiently compared and contrasted with results from existing published literature, which undermines the credibility of the study's conclusions.

Response 5: We thank the Reviewer for this comment. We acknowledge that mechanistic explanations for the species-specific effects of PEF on chlorophyll content are limited in the manuscript. However, it is important to note that, to our knowledge, there are very few studies addressing chlorophylls and carotenoids specifically after PEF priming of seeds. Current literature, as far as we know, does not provide many detailed mechanistic insights that could explain these differences We revised and added a paragraph on possible mechanisms impacted that could hypothesize the motives behind these, while emphasizing the novelty of the study and the need for further mechanistic research. (Lines 501-514, highlighted)

Comment 6: The caption for Figure 1 (Chlorophyll and Carotenoid content) states "Statistical comparisons were conducted within each colour group," which is unclear. It should explicitly state that comparisons were made between different treatment groups within each species.

Response 6: We thank the Reviewer for this comment. Indeed, there was an error in the original caption, which has now been revised to clearly state that statistical comparisons were made between different treatment groups within each species. Alterations are highlighted in the caption.

Comment 7: The Sensory Analysis section (3.3) notes that differences between groups were not statistically significant, yet considerable text is devoted to describing minor numerical variations on the radar charts. Given the lack of statistical significance and likely minimal practical relevance of these differences, this section should be condensed, focusing on the primary conclusion of "no significant effect."

Response 7: We thank the Reviewer for this comment. We agree that the overall conclusion of the sensory analysis is that no statistically significant differences were found between treatments. However, we chose to briefly describe the minor variations in order to give a complete picture of the sensory trends and to aid in the interpretation of possible tendencies which, while not statistically significant, may still pose practical or perceptual interest for future studies investigating sensory changes induced by PEF electropriming in microgreens.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents the study of the effect of PEF on germination parameters and the composition of metabolites and nutrients in the species. (Beta vulgaris L.), arugula (Eruca vesicaria), and basil (Ocimum basilicum L.). The results show a favorable effect on germination performance and varying effects on nutrient components. This work offers a novel alternative for seed priming treatments with potential applications in propagation programs or the production of sprouts as food.

The work is clearly written, and the protocols used are appropriate and concise for evaluating the response variables.

One of the main limitations I see in this work is the lack of a discussion as part of the standard format of a publication, in which previous reports can be compared with those obtained and, especially, an interpretation of the results can be offered. For example, increased root length can be associated with various nutrient and environmental factors, or variations in growth regulators; however, the required analysis and discussion are lacking.

Additionally, the following suggestions are made:
Page 4, line 134. I suggest that you provide an explanation or justification for Why aren't the PEFs the same for each species evaluated?
Page 7, Lines 283-287. Was confirmation made that the FT-NIR curves are applicable to the samples evaluated in this study?

Author Response

Comment 1: The manuscript presents the study of the effect of PEF on germination parameters and the composition of metabolites and nutrients in the species. (Beta vulgaris L.), arugula (Eruca vesicaria), and basil (Ocimum basilicum L.). The results show a favorable effect on germination performance and varying effects on nutrient components. This work offers a novel alternative for seed priming treatments with potential applications in propagation programs or the production of sprouts as food.

The work is clearly written, and the protocols used are appropriate and concise for evaluating the response variables.

Response 1: We sincerely thank the reviewer for this valuable comment. We agree that a more detailed discussion is essential to interpret our findings, therefore we now discuss possible physiological explanations for the observed increase in radicle length and germination performance (Lines 449-476 - Highlighted in Red).

Comment 2: Additionally, the following suggestions are made:
Page 4, line 134. I suggest that you provide an explanation or justification for Why aren't the PEFs the same for each species evaluated?

Response 2: Dear Reviewer, we thank you for this comment. The variation in water volume and electrode area was only to accommodate the different seed sizes and ensure full immersion between the electrodes. The PEF input parameters were identical for all species Regarding the differences in current (A), energy per pulse, and total energy (Ws) between protocols and species, these occur because those are output values not defined by the equipment, but dependent on the electrical conductivity and homogeneity of the load, pulse voltage, electrode area, and distance between the electrodes. The input values set on the equipment (voltage, frequency, pulse width, number, and shape) and electrode distance (d) were consistent across all species.

Comment 3: Page 7, Lines 283-287. Was confirmation made that the FT-NIR curves are applicable to the samples evaluated in this study?

Response 3: Dear Reviewer, we appreciate your comment. The FT-NIR output was obtained using the instrument’s validated calibration models, which had been previously tested for plant-based materials with similar composition.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The reviewer acknowledges the authors' detailed point-by-point responses to the review comments. The revised manuscript shows significant improvement compared to the original version and essentially meets the journal's publishing standards.

Author Response

The authors extend their sincere thanks to the reviewers for their valuable time and constructive suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed the suggestions for correcting the manuscript.

Author Response

The authors extend their sincere thanks to the reviewers for their valuable time and constructive suggestions.