Cryopreservation of Lavender Trumpet Tree (Handroanthus impetiginosus) Seeds

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Specific Comments and Methodological Suggestions:

Use of Vitrification Solutions

The use of plant vitrification solutions (PVS) for the cryopreservation of large seeds, like those of Handroanthus impetiginosus, may not be necessary and could even be counterproductive. These seeds demonstrate tolerance to drying, and with a water content of 6.5% (as reported in this study), they are likely to vitrify at high cooling rates without requiring additional cryoprotectants. Large seeds face challenges in fully absorbing vitrification solutions due to their thick, impermeable seed coats, which limit penetration, as well as their low metabolic activity, which reduces osmotic uptake. The diverse internal structures within seeds can also lead to uneven solution absorption.

If the authors choose to continue using PVS in further studies, a different approach may be warranted. Non-penetrating cryoprotectants work by dehydrating cells, drawing water out to minimize external ice formation, whereas penetrating cryoprotectants replace intracellular water to prevent ice formation within cells and stabilize them in a vitrified, glass-like state. Combined solutions utilize both mechanisms, removing water externally and internally. Given this, a more effective method might involve forgoing initial drying and instead using PVS solutions directly for dehydration, which could improve cryoprotection without the need for excessive pre-treatment.

Role of Loading Solution

In standard vitrification protocols, a loading solution is often used prior to PVS to gradually dehydrate tissues with high water content and prepare them for concentrated PVS solutions. However, in this study, the seeds’ water content was approximately ten times lower than the water content of the loading solution. Consequently, soaking the seeds in a loading solution for an hour may have resulted in hydration rather than dehydration, contradicting the intended purpose of the pre-treatment. This protocol adaptation does not align with the needs of seeds with low water content and may explain some of the inconsistencies observed in germination outcomes.

Warming Stage

Standard protocols that use loading solutions and PVS typically employ an unloading solution to gradually remove highly concentrated cryoprotectants, preventing osmotic shock and cellular damage. Here, however, distilled water was applied directly, which might be inappropriate. But I think this was not so critical for seeds, as it would be critical for cell suspensions or meristem tissues.

Analysis of Results

The study lacks sufficient analysis of the observed results, particularly regarding the six processing types. A deeper exploration of why certain cryopreservation protocols were less effective would enrich the discussion. The comments above could be used to better explain the unsuccessful outcomes with PVS solutions. Understanding the mechanisms behind these results would provide clearer guidance for optimizing future cryopreservation protocols for Handroanthus impetiginosus and similar species.

Specific points:

1. Title (Line 2): Suggest “Cryopreservation of Lavender Trumpet Tree (Handroanthus impetiginosus) Seeds” instead of “in.”
2. Line 54: Replace “In addition to quality seed quality” with “In addition to ensuring seed quality” for clarity.
3. Line 71-72: “The utilization of highly concentrated and pre-cooled cryoprotective solutions, vitrification prevents…” would be clearer as “The utilization of highly concentrated and pre-cooled cryoprotective solutions in vitrification prevents…”
4. Line 85-86: “The collection took place in trees used for urban landscaping in May 2023” is better rephrased as “The collection took place in May 2023 from trees used for urban landscaping.”
5. Line 86: Change “This region has a humid subtropical climate is classified as USDA…” to “This region has a humid subtropical climate and is classified as USDA…”
6. Line 87: The comma in “The average annual precipitation is 1,230 mm” may be confusing; it would be clearer as “1230 mm” without the comma.
7. Lines 89-105 (Water Content Determination): This section is overly complicated. In the results, only the water content for the control and after one day of drying in silica gel is relevant. The phrase “The initial and final moisture contents were measured after the seeds had been placed in silica gel to desiccate for one day” implies that the initial moisture content was measured after drying, which may be misleading. The study seems to involve a single-stage drying of seeds in silica gel, with oven drying used only for calculating dry weight via formula 1. Formula 2 appears irrelevant here, as it’s typically used for high initial moisture content, requiring two-stage drying. Additionally, brackets in formula 2 are placed incorrectly, potentially leading to a negative result. “The moisture content as a percentage by Weight (fresh weight basis) is calculated…” would be clearer as “The moisture content as a percentage by fresh weight is calculated…”
8. Lines 112 and 165 (Manufacturer Information Consistency): It would improve consistency to format manufacturer information uniformly throughout. For example, Line 112 should read “(KUBTEC Scientific, Stratford, Connecticut, USA)” and Line 165 as “(model AP50, Denver Instrument Company, Denver, CO, USA)” similar to Line 169.
9. Line 131: It seems incorrect to say “they were either placed in an ultra-freezer” for the control group. It likely should read “they were not placed in an ultra-freezer” as it’s referring to the control.
10. Line 146-147: “The treatments were immersed for 120 minutes in each solution” might be clearer as “The samples were immersed for 120 minutes in each solution.”
11. Line 157-158: “The acrylic boxes and trays were growth chamber…” is unclear and would be clearer as “The acrylic boxes and trays were placed in a growth chamber…”
12. Line 226: It seems “severed dehydration” should be “severe dehydration.”

Comments for author File: Comments.pdf

Author Response

Comment 1: Methodological Suggestions (Use of Vitrification Solutions; Role of Loading Solution; Analysis of Results)

Response 1: Thank you for your valuable suggestions. We have addressed them point by point in the discussion section. The corresponding revisions can be found on lines 385-393, 417-436, and 460-463, and all changes have been highlighted in red font for easy reference.

 

Comment 2: Methodological Suggestions (Warming Stage)

Response 2: Thank you for your comment. We agree regarding the use of distilled water in the unloading stage. While standard protocols typically employ unloading solutions to gradually remove concentrated cryoprotectants and prevent osmotic shock, we did not include a detailed discussion of this in the manuscript. This is because we believe that, for seeds, the effect is not as critical as it would be for more sensitive plant tissues, such as cell suspensions or meristems.

 

Comment 3: Specific Points

Response 3: Thank you for your suggestions on the main text. We agree with your recommendations and have made the appropriate revisions line by line. All changes are highlighted in red font, with the specific lines marked as per your suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

It was very important to conserved seeds material, but there are few points needed to be consided when design the experiment:

1. Cryoprotectent. The cryoprotectent used to reduce the water content, however MC here is 12%, thus no need to apply cryoprotectent, otherwise , it will increase water content and bring poison to the cells.

2. X ray will damage genetic level of the cell, for the thretend species, it may not a good idea to use X ray before stored the seeds.

Thus, the paper didn't quite logical design for the experimental, and  it would be great if the author could reconsider the scientific question and use the proper method to answer it.

Author Response

Comment 1: Cryoprotectent. The cryoprotectent used to reduce the water content, however MC here is 12%, thus no need to apply cryoprotectent, otherwise, it will increase water content and bring poison to the cells.

Response 1: Thank you for your insightful comment. We agree with your observation regarding the use of cryoprotectants. With the moisture content (MC) of the seeds at 12%, there is no need for cryoprotectants, as they could increase water content and potentially cause harm to the cells.

To provide better clarification, we have expanded the discussion in the manuscript regarding the seed moisture content and its impact on the effectiveness of cryopreservation solutions. Additionally, we suggest that for seeds with higher moisture content, the response to cryoprotectant solutions may differ. In such cases, it is advisable to avoid the use of these solutions, particularly when pre-drying is applied.

We hope this addition enhances the manuscript and addresses your concerns. Thank you once again for your valuable feedback.

Comment 2: X ray will damage genetic level of the cell, for the thretend species, it may not a good idea to use X ray before stored the seeds.

Response 2: Thank you for the reviewer’s valuable feedback. I would like to clarify that the main focus of my study was not to directly evaluate the potential genetic damage caused by X-ray radiation, but rather to explore X-ray imaging as a tool for assessing seed quality in a broader context of cryopreservation research.

In the study, X-ray imaging was employed as a non-invasive method to assess internal seed structures and to gain insights into the viability and preservation potential of the seeds before and after cryopreservation treatments. I fully acknowledge the concerns regarding radiation damage to the genetic material of seeds, as even low doses of X-rays can cause DNA strand breaks and mutations. However, it is important to emphasize that the use of X-ray imaging in this context was limited to low-dose exposures designed to minimize such risks. The typical doses used in imaging for seed viability studies tend to be relatively low compared to those used in mutation studies or other high-damage radiation treatments. In fact, several studies have employed low-dose X-ray imaging for seed analysis without significantly compromising seed viability or genetic integrity (Udroiu & Sgura, 2023). Finally, the use of X-ray imaging in terms of conservation is already employed for several species, including threatened species, as an initial quality assessment to ensure purity and seed quantity, as well as a non-destructive quality assessment over time during long term storage (Liu et al., 2020) (This section in bold was added to discussion line 363-366). Altogether, these studies suggest that X-ray imaging can be a useful tool when used cautiously.

We would like to emphasize that it was never the primary aim of this paper to delve into the long-term effects of X-ray exposure on the genetic material of the seeds, but rather to explore imaging techniques that could assess seed quality in terms of morphological damage and inform the design of cryopreservation protocols. The potential for DNA damage, though valid, is not central to this study, and I agree that further research into this aspect would be essential for future studies that aim to understand the broader implications of X-ray usage on genetic material.

Thank you again for your feedback, and I am happy to reconsider the design of future experiments to further address these concerns.

Udroiu, I.; Sgura, A. X-ray and DNA Damage: Limitations of the Dose as a Parameter for In Vitro Studies. Int. J. Mol. Sci. 2023, 24, 16643. https://doi.org/10.3390/ijms242316643

Liu, U., Cossu, T. A., Davies, R. M., Forest, F., Dickie, J. B., & Breman, E. Conserving orthodox seeds of globally threatened plants ex situ in the Millennium Seed Bank, Royal Botanic Gardens, Kew, UK: The status of seed collections. Biodiversity and Conservation, 2020, 29(11-12), 2901-2949. https://doi.org/10.1007/s10531-020-02005-6

Reviewer 3 Report

Comments and Suggestions for Authors

The paper represents a relevant and innovative contribution to the topic. The methods are clearly described and as well as  the data obtained are clearly described and discussed. The discussion is accurately performed taking into consideration previously important manuscripts on the topic in other species.

Author Response

Dear Reviewer,

Thank you for your feedback and for considering our manuscript. We believe this topic aligns well with the scope of Horticulturae and contributes valuable insights into the potential of non-invasive X-ray imaging for seed evaluation and cryopreservation techniques.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks the author to explain in the text to explain my concerning questions. As fo the X-ray, for the long term storage of such important species, not recommended.