In Vitro and Cryobiotechnology Approaches to Safeguard Lupinus rivularis Douglas ex Lindl., an Endangered Plant in Canada
Round 1
Reviewer 1 Report
Dear authors,
Your manuscript seems really interesting and is very well written. I have made several comments that I hope you find them useful. My major concern lies with the type of test you used and I think that if you reframe your aim in order to highlight the importance of cryopreservation for any species facing extinction, your work might gain more readers and have a greater impact.
Comments for author File: 
Comments.pdf
Author Response
Your manuscript seems really interesting and is very well written. I have made several comments that I hope you find them useful. My major concern lies with the type of test you used and I think that if you reframe your aim in order to highlight the importance of cryopreservation for any species facing extinction, your work might gain more readers and have a greater impact.
Line 78-80: The Introduction section flows smoothly, yet I haven't grasped what's so special about Lupinus rivularis compared to other plants that might be Critically Endangered under the IUCN Criteria. Do you aim for this manuscript to serve as a basis for CPR and chose Lupinus rivularis as a model? Could you please elaborate more on the reasons why you chose this specific endangered species and not others that might be facing imminent extinction? Do you aim to alert conservationists regarding a procedure that might be cost-effective in protecting imperiled species compared to other practices? I think that by doing so, you might upscale the importance of your work.
Currently, few alternate conservation and preservation plans for the species in the classified range of extinction process or staged at extinction, exist. Relevant to such utmost probability, here, we demonstrate validation of the conservation, propagation and redistribution (CPR) model using Lupinus rivularis, a plant species rated as endangered in Canada. We have provided evidence of the usefulness and practical application of micropropagation and cryopreservation technologies for species recovery. The study also provides an integrated approaches in plant conservation, propagation and redistribution and demonstrates potential for saving other endangered plant species.
Also, species such as Lupinus rivularis can offer agricultural and medicinal benefits which further emphasizes the need for conservation of endangered species. These aspects have been included in the text.
Line 138: and maximum?
Sentences have been edited by adding maximum period (48 h)
Line 154 and 163: Reference format does not look as it should
Text has been modified for reference citation.
Line 183: DMRT raises Type I error rates, following the Newman-Keuls procedure. Could you please consider another approach, such as the Duncan-Waller test or Tukey's range test?
Text and figures have been modified by analysing the data as per Tukey test.
Line 202, 235, 260 (figure 1, 3 and 4): Could you please consider changing the resolution of this figure? It seems coarse and difficult to read. Could you please also consider changing the color palette, as the colors seem more or less the same to color-blinded people?
Figure 1 have been modified with colored bars and brightness. Figure 3 has been replaced by table 1 as per other reviewer’s suggestion.
Line 206: replication
Total four replications, already mentioned in line 205.
Line 207: Also please state here why panel C has data for seed lot 1 (you mention it in the text, but it could be useful if the reader reads it here as well).
Figure legend has been modified by adding information.
Line 225: Could you please explain why you chose not to share these data?
Data has been included in table 1 (Fig 3 has been changed to Table 1).
Line 285: Please consider making the table lines black
Table has been modified with black lines.
Line 287: This is confusing. Why not just state which means are s.s. different? As I mentioned earlier, please consider using another, more appropriate test for your analysis.
Text and figures have been modified by analysing the data as per Tukey’s HSD test.
Line 294: Could you please explain why you prefer not to share these data?
As we mentioned in the text that all shoot tips survived in the cryoprotectant solution without liquid nitrogen treatment. Text has been modified by adding 100% survival.
Line 304: Please see my first comment regarding the importance of your work
Please see the justification as above.
Line 357: This is an entirely different "beast" - Quercus spp. are shrubs or trees, while Lupinus spp. are herbs.
We have included this reference to show the importance of explant selection and their in vitro responses.
Line 360-361: Please write the binomials for guava and hazelnut.
Text has been modified
Line 404: This is why you should state the maximum time spent in liquid nitrogen for the seeds used in your experiments
Text has been modified by adding time interval.
Line 414: change etc with among others - but please state some more parameters
Text has been modified by adding more parameters
Reviewer 2 Report
Dear authors,
congratulations for the manuscript.
I think that you could improve the reliability of data statistical analysis using Students t test. This is very important, since nowdays this (or Tukey HSD. depending on 'n ' number) is the basic statistical method for one-way ANOVA.
The Students t test (and even more Tukey HSD) shows more-detailed results compared with Duncan.
You could also edit a data-table instead of Figure 3, including also percenatages data. It is very important to present percentages of shoot formation in in vitro experiments.
As regards other corrections, there is a short list:
- Line 3: Use italics for species - check everywhere (i.e. 205 etc)
- Line 16: Use italics for 'ex situ' and 'in vitro'
- Line 21: Canada. (not Canada .)
- Line 21: Use italics for 'in vitro'. Check everywhere in the paper for similar phrases
- Line 29: Check 'spaces'
- Line 32,33: Use alphabetical order
- Line 46-58: You could use also the next reference: https://dx.doi.org/10.2305/IUCN.UK.2012.RLTS.T19891938A20100330.en
- Line 72-73: Correct the references (use numbers)
- Line 90: Check spaces
- Line 95: Correct to: 121 oC
- Line 126: Correct to 118 kPa
- Line 122: What is the number of replications?
- Line 132: Check spaces before units
- Line 140 and 144: Check spaces before units
- Line 207: delete word 'value' (also for 240, 265, 290)
- Line 215: Use smaller characters for A/B/C/D
- Line 455: You have to check the references style everywhere.
Author Response
I think that you could improve the reliability of data statistical analysis using Students t test. This is very important, since nowdays this (or Tukey HSD. depending on 'n ' number) is the basic statistical method for one-way ANOVA.
The Students t test (and even more Tukey HSD) shows more-detailed results compared with Duncan.
Text and figures have been modified by analysing the data as per Tukey test.
You could also edit a data-table instead of Figure 3, including also percenatages data. It is very important to present percentages of shoot formation in in vitro experiments.
Figure 3 changed to table and incorporated percentage data. Text has been updated.
As regards other corrections, there is a short list:
Line 3: Use italics for species - check everywhere (i.e. 205 etc) Sentences have been edited.
Line 16: Use italics for 'ex situ' and 'in vitro' Text has been updated.
Line 21: Canada. (not Canada .) Text has been updated.
Line 21: Use italics for 'in vitro'. Check everywhere in the paper for similar phrases Text has been updated.
Line 29: Check 'spaces' Text has been updated.
Line 32,33: Use alphabetical order Text has been updated.
Line 46-58: You could use also the next reference: https://dx.doi.org/10.2305/IUCN.UK.2012.RLTS.T19891938A20100330.en
We have cited original reference COSEWIC 202 and same references cited in the IUCN database.
Line 72-73: Correct the references (use numbers) Text has been updated
Line 90: Check spaces Text has been updated.
Line 95: Correct to: 121 oC Text has been updated.
Line 126: Correct to 118 kPa Text has been updated.
Line 122: What is the number of replications? A sentence has been added in the text.
Line 132: Check spaces before units Text has been updated.
Line 140 and 144: Check spaces before units Text has been updated.
Line 207: delete word 'value' (also for 240, 265, 290) Text has been updated.
Line 215: Use smaller characters for A/B/C/D Figure letters have been changed
Line 455: You have to check the references style everywhere. Text has been modified for two references and verified other references as per the author’s instruction.
Reviewer 3 Report
This manuscript describes efficient protocols for propagation and cryopreservation of Lupinus rivularis, endangered species whose germplasm requires conservation. The experiments were well designed, and results are encouraging. The discussion is very well argued.
Followings are few points for consideration:
Line 95: °C, ° is missing
Line 96: 118 kPa (space)
Line 123, 237, 245: simbol is missing (micro M) (It was not possible to paste it here as well)
Line 126: It would be desirable to write from which previous treatment (BA concentration) shoots were taken for rooting
Line 162: 1.5 x 1.5 (space)
Line 205: put the latin name in italic
Line 263-265: unnecessary clarification - that in figures no. 4 does not exist (probably mistakenly copied from the description of figure 1)
Author Response
This manuscript describes efficient protocols for propagation and cryopreservation of Lupinus rivularis, endangered species whose germplasm requires conservation. The experiments were well designed, and results are encouraging. The discussion is very well argued.
Followings are few points for consideration:
- Line 95: °C, ° is missing Text has been updated.
- Line 96: 118 kPa (space) Text has been updated.
- Line 123, 237, 245: simbol is missing (micro M) (It was not possible to paste it here as well) Text has been updated.
- Line 126: It would be desirable to write from which previous treatment (BA concentration) shoots were taken for rooting Text has been updated.
- Line 162: 1.5 x 1.5 (space) Text has been updated.
- Line 205: put the latin name in italic Text has been updated.
- Line 263-265: unnecessary clarification - that in figures no. 4 does not exist (probably mistakenly copied from the description of figure 1) Text has been updated.
