Seed Germination Trials and Ex Situ Conservation of Local Prioritized Endemic Plants of Crete (Greece) with Commercial Interest
, Georgios Tsoktouridis, Eleni Maloupa and Katerina Grigoriadou *
Round 1
Reviewer 1 Report
The manuscript is focused on the propagation, through seed germination, of nine species endemic to Crete (Greece). It is important the development of propagation protocols and the analysis performed in terms of the potential use of these threatened species.
However, some major corrections should be performed in order to make clear some specific points and to improve the quality of this paper.
Introduction
Introduction is fine, although three points should be considered:
1) It is not necessary to include in this section the description of each species. This is already described in Material and Methods (lines 85-97).
2) Considering this manuscript is focused on seed germination, information about seed germination barriers and how to overcome them is missing. According to the geographical distribution of the species studied, one can assume that these seeds present some type of dormancy and need stratification. This physiological phenomenon should be introduced in this section, as well as the different type of dormancies (i.e. physiological / morphological dormancy) particularly present in species native to Mediterranean climate areas.
3) Considering the treatments with GA3 application described in Material and Methods, its effect on seed germination (i.e. physiological effect over dormancy, interaction with ABA) should be included in this section.
Material and Methods
This section should be improved as some important information is missing and/or not clear:
1) On Figures 1 to 4, each single picture should be identified with a letter and then specifically described in the caption of the figure.
2) For the first in vivo sowing, these seeds should have been soaked 24 h in dH2O in order to perform an appropriate comparison with the treatments using GA3 (lines 179-181).
3) Can you really compare these two in vivo sowing experiments (first and second) considering they were performed at different time of the year, with different 'age' (i.e. different physiological state, level of dormancy, humidity, etc.) of the seeds.
4) A control should have been included (0 ppm GA3) (line 185).
5) Measurements should be accounted as ‘days after germination’ and not as ‘15th, 30th , etc. day’.
6) Was there any criteria (i.e. phenological stage or plant height) for the seedling transplantation to larger volume pots? (line 194).
Results
This section should consider the following comments:
1) As previously suggested, each single picture should be identified with a letter and then specifically described in the caption of the figure (Figures 6 to 8).
2) This is Material and Methods, not results (lines 230 to 237).
3) This is too vague. It should be more precise (line 242).
4) Table 2:
a. ‘What is the unit measure for ‘Germination onset day’? number of days? This should be clearer on the table. If so how ‘germination day’ is defined? As the day when the first seed was germinated?
b. 15th , 30th , etc. should be ‘days after sowing’.
c. What is the difference between 0 ppm GA3 and 24 h in H2O? (i.e. Treatment for Draba cretica). This should be explained in Materials and methods.
5) Why the authors assume this is taxon-dependant? In any case, interpretation of the results should be included in the Discussion section (line 257).
6) What is t50? Definition should be given (line 263).
7) Table 4. Why this data is not together with table 2? Shouldn't it be only one table? Why different evaluations were taken for the same treatments? If this is not a mistake, it should be clearly justified in the Material and Methods section. I.e. Table 2 shows germination rate while Table 4 shows t50 values.
8) There is no methodology described that is somehow associated with the evaluation of feasibility (line 315).
9) This is discussion, not results (line 344-351).
10) Table 5. Criteria, level (I, II and II) and each ‘level of interest’ should be fully explained in Material and Methods. I would not mix evaluation if feasibility with data coming from seed germination.
Discussion
This section is well structured and rich in terms of information and level of discussion.
Conclusions
This section is a bit to long. The author should focus more on conclusions coming from the results of this particular study. Some specific comments:
1) By ‘lethargic’ maybe you mean dormancy? (line 587).
2) This is result, not conclusion (lines 594-600)
Author Response
Review Manuscript code number: Seeds-1946029
Manuscript title: Seed germination trials and ex-situ conservation of local prioritized endemic plants of Crete (Greece) with commercial interest
Response letter to reviewers’ comments
Reviewer #1
The manuscript is focused on the propagation, through seed germination, of nine species endemic to Crete (Greece). It is important the development of propagation protocols and the analysis performed in terms of the potential use of these threatened species. However, some major corrections should be performed in order to make clear some specific points and to improve the quality of this paper.
Introduction
Introduction is fine, although three points should be considered:
1) It is not necessary to include in this section the description of each species. This is already described in Material and Methods (lines 85-97).
Authors’ response: We have followed the reviewer’s suggestion and we have shortened the text omitting detailed information regarding extinction risk, commercial interest and family for each taxon. Therefore, the text was re-written including only the scientific and common names of the nine endemic plants of Crete under study. The new (modified) text in the revised manuscript has been highlighted with green color (see page 3, lines 115-121).
2) Considering this manuscript is focused on seed germination, information about seed germination barriers and how to overcome them is missing. According to the geographical distribution of the species studied, one can assume that these seeds present some type of dormancy and need stratification. This physiological phenomenon should be introduced in this section, as well as the different type of dormancies (i.e. physiological / morphological dormancy) particularly present in species native to Mediterranean climate areas.
Authors’ response: We have followed the reviewer’s suggestion and and the requested information was provided. We have additionally included four (4) new citations (Geneve 2005, Hilhorst 2006, Finch-Savage and Leubner-Metzger 2006, Liu and Hu 2018) into the text and in the reference list, thus changing the original enumeration of almost all references. In the revised manuscript, the newly inserted text is green-labelled and all amendments are indicated with track changes (see pages 2-3, lines 80-108).
3) Considering the treatments with GA3 application described in Material and Methods, its effect on seed germination (i.e. physiological effect over dormancy, interaction with ABA) should be included in this section.
Authors’ response: We have followed the reviewer’s suggestion and the requested information was provided. In the revised manuscript, the newly inserted text is green-labelled and all amendments are indicated with track changes (see pages 2-3, lines 80-108).
Material and Methods
This section should be improved as some important information is missing and/or not clear:
1) On Figures 1 to 4, each single picture should be identified with a letter and then specifically described in the caption of the figure.
Authors’ response: We have followed the reviewer’s advice and we have identified all photos in Figures 1-4 with respective letters which are described in the respective captures of the figures (see pages 6-8, changes in green colour).
2) For the first in vivo sowing, these seeds should have been soaked 24 h in dH2O in order to perform an appropriate comparison with the treatments using GA3 (lines 179-181).
Authors’ response: We would like to thank the reviewer for this comment which gave us the opportunity to better describe the approach followed in this investigation. Following the reviewer’s suggestion, we have revised the section 2.3 (see track changes and green highlighted texts, page 9).
3) Can you really compare these two in vivo sowing experiments (first and second) considering they were performed at different time of the year, with different 'age' (i.e. different physiological state, level of dormancy, humidity, etc.) of the seeds.
Authors’ response: We agree with the reviewer’s comment. Due to different seed storage conditions, different pre-treatment regimes, and different number of tested taxa between the two sowing seasons of the year, no further comparisons were made. This has now been clearly indicated in the revised version of the manuscript (see pages 9-10, lines 249-258).
4) A control should have been included (0 ppm GA3) (line 185).
Authors’ response: For Calamintha cretica, Onopordum bracteatum subsp. creticum and Origanum microphyllum, the 24h immersion of seeds in dH2O (same or equivalent to 24h in 0 ppm GA3 solution) served as the control treatment to the increased concentrations of GA3 (50 and 250 ppm for O. bracteatum subsp. creticum, but only 50 ppm for C. cretica and O. microphyllum), thus 24h in 0 ppm GA3 solution is equivalent to 24h in dH2O (same pretreatment). This has now been clearly indicated in the revised version of the manuscript (see page 9, lines 221-226).
5) Measurements should be accounted as ‘days after germination’ and not as ‘15th, 30th, etc. day’.
Authors’ response: We have corrected this throughout the manuscript (see scattered green-labelled changes across the revised manuscript’s texts).
6) Was there any criteria (i.e. phenological stage or plant height) for the seedling transplantation to larger volume pots? (line 194).
Authors’ response: We have followed the reviewer’s suggestion and in the revised manuscript we have amended this criterion (see page 10, lines 266-270). Τhe criterion for transplanting the plants into larger volume pots was the first emergence of the root system out of the container, thus indicating symptoms of intolerance and non-symmetrical growth of the underground part compared to the above ground part.
Results
This section should consider the following comments:
1) As previously suggested, each single picture should be identified with a letter and then specifically described in the caption of the figure (Figures 6 to 8).
Authors’ response: We have followed the reviewer’s advice and we have identified all photos in Figures 1-4 with respective letters which are described in the respective captures of the figures (see changes in green colour).
2) This is Material and Methods, not results (lines 230 to 237).
Authors’ response: Following the reviewer’s suggestion, the indicated text was transferred from Results to M&M section, subsection “2.3. Seed germination trials”, 2nd paragraph (see revised manuscript, page 9, lines 221-222, changes in green colour), after being re-phrased for better clarity, as requested.
3) This is too vague. It should be more precise (line 242).
Authors’ response: We would like to thank the reviewer for this comment which gave us the opportunity to improve our manuscript. The initial sentence was re-written to better clarity this issue and new text was added to describing accurately the results supporting the afore-mentioned initial sentence (see page 15, lines 332-349). One may now read as follows: “According to Table 4 (combined overview of Table 2 and Table 3), sowing season of the year (autumn, spring), cold and non-previous storage of seeds, 24 h seed pretreatment prior to sowing in dH2O and/or GA3 solutions, and the taxon’s identity seem to be important factors that can affect the germination ability of the seeds either positively, negatively or not at all. In specific, there was acceleration in the germination onset from 13 to 9 days after sowing and higher germination percentages for Calamintha cretica seeds sown in spring after a 24 h pretreatment with 50 ppm GA3 compared to autumn sowing under the same pretreatment condition (24 h in 50 ppm GA3) or in spring after a 24 h pretreatment in dH2O. Moreover, higher germination percentages were obtained for Dianthus fruticosus subsp. creticus and Draba cretica seeds sown in autumn compared to those sown in spring. In the case of pre-treated seeds of Origanum microphyllum with 50 ppm GA3 for 24 h, the initiation of germination (in days) occurred earlier during autumn sowing than during spring (Table 4). The germination percentage of Onopordum bracteatum subsp. creticum seeds pre-treated for 24 h in dH2O in spring (18%) was 12-fold higher than that (1.5%) obtained in the autumn sowing, while the pre-treatment of spring-sown seeds with different concentrations of GA3 solutions (0, 50, 250 ppm) did not manage to raise considerably the germination percentages (23-25%) of the control treatment (18%) (24 h in dH2O or 0 ppm GA3) (Table 4, Figure 9).”
4) Table 2:
- ‘What is the unit measure for ‘Germination onset day’? number of days? This should be clearer on the table. If so how ‘germination day’ is defined? As the day when the first seed was germinated?
Authors’ response: The unit measure used for “Germination onset day” was the number of days, and the onset (i.e., initiation) of germination was defined as the day when the first seed was germinated. This statement was included in M&M section, subsection 2.3. (see page 9, lines 237-240).
- 15th, 30th , etc. should be ‘days after sowing’.
Authors’ response: We have corrected this issue throughout the manuscript (see scattered changes green labelled in the revised version of the manuscript).
- What is the difference between 0 ppm GA3 and 24 h in H2O? (i.e. Treatment for Draba cretica). This should be explained in Materials and methods.
Authors’ response: We have now clarified this in the revised version of the manuscript indicating that “24h in 0 ppm GA3 solution is equivalent to 24h in dH2O (same pretreatment)”. More detailed description and better explanation is now provided in M&M section as suggested (see green labelled text in section 2.3, page 4, lines 218-219 & 221-222).
5) Why the authors assume this is taxon-dependent? In any case, interpretation of the results should be included in the Discussion section (line 257).
Authors’ response: We have followed the reviewer’s suggestion. The whole sentence was deleted from Results section and transferred after re-phrasing in the Discussion for better interpretation of the findings of this study.
6) What is t50? Definition should be given (line 263).
Authors’ response: In M&M section the following definition was given for t50: “In addition, the median germination time (t50) was calculated according to the formula by Farooq et al. (2005) as the time needed (in days) to reach 50% of the final/ maximum germination (Tables 2-3). (see green labelled text in section 2.3, page 9, lines 235-237).
7) Table 4. Why this data is not together with table 2? Shouldn't it be only one table? Why different evaluations were taken for the same treatments? If this is not a mistake, it should be clearly justified in the Material and Methods section. I.e. Table 2 shows germination rate while Table 4 shows t50 values.
Authors’ response: These experiments cannot be directly compared considering that the two in vivo sowing experiments (first and second) were performed at different times (autumn and spring) using seeds of different 'age' (i.e., different physiological state, level of dormancy, humidity, etc.). Seeds of the first sowing trial in autumn received no previous cold storage or 24 h pre-treatment (nine taxa) whereas seeds of the second trial in spring were kept for 80 days in a cold chamber (4-5oC, RH <5%) (six taxa) and pre-treated for 24 h with dH2O and/ or GA3 solutions, thus with different storage conditions and different pre-treatment regimes. The data presented in Tables 2, 3 and 4 cannot be put together in a single Table, due to the reasons described above. In the revised manuscript, however, we have re-arranged the order of appearance of these Tables for better clarity in an attempt to provide concise understanding of the text in the Results section. In specific, Table 2 was enumerated as Table 4, Table 3 remained with the same enumeration (no changes occurred) whereas Table 4 was re-enumerated as Table 2. In the revised version of our manuscript, Table 2 includes germination percentages and t50 values for nine endemic Cretan taxa sown in the autumn without previous cold storage or pretreatment, Table 3 includes germination percentages and t50 values for six endemic Cretan taxa sown in the spring after 80 days of cold storage and 24h pretreatment in dH2O or in GA3 solutions (taxon-dependent) while Table 4 presents a combined overview of both in vivo germinations trials (Tables 2-3) including also the “germination onset day” column.
8) There is no methodology described that is somehow associated with the evaluation of feasibility (line 315).
Authors’ response: We have followed the reviewer’s advice and we have preferred to move this part into the discussion section (see revised manuscript, page 21, lines 584-624, changes in green colour). All methodological issues are presented in detail in previously published papers (Krigas et al. 2021, Bougou et al 2021, Libiad et al. 2021).
9) This is discussion, not results (line 344-351).
Authors’ response: We have followed the reviewer’s advice and we have preferred to move this part into the discussion section (see page 21, lines 617-624, changes in green colour).
10) Table 5. Criteria, level (I, II and II) and each ‘level of interest’ should be fully explained in Material and Methods. I would not mix evaluation if feasibility with data coming from seed germination.
Authors’ response: We have followed the reviewer’s advice and we have preferred to move this part into the discussion section (see Table 5, pages 21-22, lines 627-635, changes in green colour). All methodological issues are presented in detail in previously published papers (Krigas et al. 2021 Bougou et al 2021, Libiad et al. 2021) (see [4-6], in the reference list).
Conclusions
This section is a bit too long. The author should focus more on conclusions coming from the results of this particular study. Some specific comments:
1) By ‘lethargic’ maybe you mean dormancy? (line 587).
Authors’ response: To address the reviewer’s comment, the phrase “appear to be lethargic” was replaced by “appear to have dormancy”. (see revised manuscript, page 23,line 644, changes in green colour).
2) This is result, not conclusion (lines 594-600).
Authors’ response: This text was deleted from the conclusions section and was transferred in the Results as the last paragraph of this section, thus shortening the extent of the conclusions section as kindly suggested (see green-labelled text, page 16, lines 350-355 in the revised manuscript).
Author Response File: 
Author Response.doc
Reviewer 2 Report
see attachment
Comments for author File: 
Comments.pdf
Author Response
Review Manuscript code number: Seeds-1946029
Manuscript title: Seed germination trials and ex-situ conservation of local prioritized endemic plants of Crete (Greece) with commercial interest
Response letter to reviewers’ comments
Reviewer #2
The manuscript describes studies of different germination tests and germination methods of nine endemic species of Crete. However, in the introduction as well as in the discussion the focus and topics are much broader like “sexual propagation”, “ex-situ conservation”, “commercial interest”. All these topics are not studied in the paper. The paper should focus on germination only.
Authors’ response: Since reviewer 1 suggested to move these parts to the discussion section, we have addressed in a combined way the advice offered by both reviewers and we have also accepted the comments of reviewer 2. Therefore, we have preferred to move the section 3.2. from the results into the discussion section (see changes in the revised manuscript with green colour, pages 21-22). All methodological issues and relevant discussion are presented in detail in previously published papers (Krigas et al. 2021, Bougou et al 2021, Libiad et al. 2021) and are therefore omitted. The section 4.2 in the discussion section is now clearly focused on and discuss how the seed germination results obtained in this study may actually contribute to the upgrading of the feasibility and readiness timescale evaluations for the sustainable exploitation of the targeted taxa.
Material-methods:
Which order do the species have, not alphabetical but why this order?
Authors’ response: In all the Tables presented in the Results section (Tables 1-5), all plant taxa are listed alphabetically based on their scientific name. Only in Figures (Figures 1-9) different plant taxa are grouped to closely related families; this was also applied for less space-covering. However, we have re-grouped the studied taxa into plants with separate petals and plants with more or less united petals in which the taxa are arranged alphabetically within their families. All the changes in section 2.1 are indicated with green color.
Seed collections: Which protocol was used; how many seeds were collected?
Authors’ response: During the seed collections in the wild we have used a customized protocol based on: (i) international guidelines such as those published from Kew, (ii) Domestic prioritization schemes suggesting massive seed harvesting in no more than 1% of the extant individuals and selective and careful harvesting form less than 10% of the wild-growing individuals per locality, (iii) Estimated accessibility combined with security in reaching the wild habitats (especially relevant for rock-dwelling plants), and (iv) Apparent plant size (small, medium, large, extra large). Therefore, the total amount of harvested seeds varied greatly form some thousands of seeds (e.g. for the easily accessed and large perennial Petromarula pinnata with widespread populations) to only some hundreds (e.g. for the miniscule and difficult to access Draba cretica forming only small scattered stands of individuals).
Seed germination trials: Why using 60 days for germination tests, ISTA says 28 days?
Authors’ response: According to the literature, no previous reports or protocols exist for the in vivo sexual propagation of the nine Cretan endemics studied herein, thus we did not know their germination ability. For this reason we tested the course of germination process taking regular measurements of germination percentages (%) at 15-day intervals within the total 60-day experimental period after sowing (15, 30, 45 and 60 days). We extended the duration of the experimental trials up to 60 days until the maximum germination percentage to be obtained. After that, the germination percentage remained stable.
Please clarify which treatment or treatments were used for which species. This should be part of methods. Also, please explain why different methods for different species were used?
Authors’ response: All this information has been improved in the revised version of the manuscript profiting from the comments made by the reviewers (see amendments in green in section 2.3). The main purpose of this study from the beginning was not the comparison between the two sowing periods but a simple reference of germination ability for each taxon, separately for different seasons of the year.
Results: Table 2: Are the values average values of three tests? Probably, it would make sense to combine table 2 with table 3 and 4.
Authors’ response: “The in vivo seed germination procedure either in autumn or in spring took place in three single seat crates of 100 seeds each, comprising a total of 300 seeds per taxon (3 replicates x 100 seeds)”. For each taxon (regardless pretreatment and sowing period), the germination percentage (%) mean value in each 15-day interval (15, 30, 45 and 60 days) was the average of 3 germination (%) values (i.e., 3 groups x 100 seeds) of the same test and not the results of three tests. For this reason and taking simultaneously into consideration the fact that the two in vivo sowing experiments performed at different time of the year (autumn and spring), with different 'age' (i.e., different physiological state, level of dormancy, humidity, different number of taxa tested in different sowing periods, etc.), data presented in Tables 2, 3 and 4 cannot be merged/combined in a single one Table.
General statement: The paper should describe the endemic species and their germination performance what is written in the abstract. The other topics should be removed because they are not investigated.
Authors’ response: Since reviewer 1 suggested to move these parts to the discussion section, we have addressed in a combined way the advice offered by both reviewers and we have also accepted the comments of reviewer 2. Therefore, we have preferred to move the section 3.2 from the results into the discussion section (see changes in the revised manuscript with green colour). All methodological issues and relevant discussion are presented in detail in previously published papers (Krigas et al. 2021, Bougou et al 2021, Libiad et al. 2021) and are therefore omitted. The section 4.2 in the discussion section is now clearly focused on and discuss how the seed germination results obtained in this study may actually contribute to the upgrading of the feasibility and readiness timescale evaluations for the sustainable exploitation of the targeted taxa.
Reviewer 3 Report
Seed germination trials and ex-situ conservation of local priori- 2 tized endemic plants of Crete (Greece) with commercial interest 3 Virginia Sarropoulou1 , Nikos Krigas1,*, Georgios Tsoktouridis1 , Eleni Maloupa1 and Katerina Grigoriadou1,*
Author Response
Review Manuscript code number: Seeds-1946029
Manuscript title: Seed germination trials and ex-situ conservation of local prioritized endemic plants of Crete (Greece) with commercial interest
Response letter to reviewers’ comments
Reviewer #3
Are the methods adequately described? / Are the results clearly presented? - Can be improved.
Authors’ response: The description of the methodology applied in M&M section has been considerably improved and the presentation of the results was provided with enhanced clarity. In addition, section 3.2 has been transferred to the discussion part as section 4.2. In this framework, extensive re-phrasing was performed in text and re-arrangements in the order of appearance of the results, Tables, Figures and their respective captions. See also, response to reviewer #1 and reviewer #2 comments as provided above as well as in the amendments highlighted with green in the revised version of the manuscript.
Author Response File: Author Response.doc
Round 2
Reviewer 1 Report
I have no further comments
Author Response
Thank you very much for your comments and positive response
Reviewer 2 Report
The manuscript has been restructured and greatly improved.
Author Response
Thank you very much for your comments and encouraging response
