Polyploid Induction and Identification of Begonia × benariensis

Comments 1: Typographical errors and more suggestions in attachment file.

Response 1: Thank you for your suggestions for formatting changes throughout the manuscript, all of which we agree with!

On page 1, lines 27, 30, 35 and on page 2, line 91, 'BIG' is marked with a ® tag.

On page 1, line27, abbreviations cancelled on the front (B.→Begonia).

The colors of the series are added on page 1, lines 32 and 33 according to the URL you posted (Highlighted in yellow).

On page 10, line 342, delete the superfluous space.

In the references section, change all those that are incorrectly capitalized and not italicized (There are traces of revisions).

Comments 2: The problem of the study is clear, but what about seeds of tetraploid plant and the possibility of using them in agriculture? In the future, desirable to establish a production program to produce hybrid Begonia × binariensis cultivars in China.

Response 2: In the introduction section, on the seeds of tetraploid plants and their potential use in agriculture had added. On page 2, lines 77-82.

On the point of establishing a production program in China in the future, this will require a lot of manpower and money to achieve, as well as the involvement of top breeders, so it is a difficult task, so it is only indicated in the last paragraph of the introduction. On page 2, lines 87-88.

Comments 3: The number of chromosomes must be added to the Begonia × benariensis hybrid, as well as for triploid and tetraploid plants.

Response 3: Chromosome numbers have been added at the corresponding positions in the abstract section. (Highlighted in yellow).

Comments 4: The Authors should rewrite the conclusions section (it's long).

Response 4: The conclusion section has been rewritten and shortened.

Comments 5: What is about flower type and it characterized in tetraploid plants? Please add these results to MS.

Response 5: Regarding the flower type of a tetraploid plant, if it is a red flower type then the tetraploid plant corresponds to a red flower type, and if it is a rose red flower type then the tetraploid plant corresponds to a rose red flower type. This is a default correspondence. Characteristics of tetraploids have been added in the conclusion section. (Highlighted in yellow)

Comments 6: The Reference section must be reformatted according to the journal.

Response 6: The references section has been reformatted according to the requirements of the journal.

4. Response to Comments on the Quality of English Language

Point 1:  Minor editing of English language required

Response 1: The English language was edited using MDPI English language editing services.

5. Additional clarifications

Response: Some references of little relevance was removed. Replaced some Chinese references with English references.

Comments 1: The processing of the manuscript has a regional character. The introduction to the issue does not include the current state of research on an international scale. Likewise, the results are not sufficiently discussed with the findings of other foreign authors.

Response 1: Begonia×benariensis germplasm resources in the hands of the German Benary company, whether for China or other countries in addition to Germany, Begonia×benariensis germplasm resources are the neck problem. This manuscript is published in this journal, which can be seen by researchers all over the world, and the experimental methods in the manuscript can be referred to for germplasm creation, which can be a reference to inspire them to break Begonia×benariensis germplasm resource problems. Industry (Benary company) is ahead of academia in both life sciences and agriculture. This manuscript is also of significance in promoting Begonia×benariensis research in the academic world.

No references on Begonia×benariensis was searched on WOS, Sci-Hub, and NCBI, and it has not been studied by any scholars on an international scale. Therefore, the geographical range has been added on page 2, line 67 (Highlighted in yellow).

As to why most of the Chinese references is used, since there are not many scholars abroad who have done the relevant research, while there are many solid factual foundations in China. For example, on the point of using colchicine for induction, whether the rate of induction is higher using the impregnation method or the mixed cultivation method has not been researched by most scholars abroad, whereas most scholars in China have applied it to different plants or fruits.

Comments 2: Unspecified area for the number of stomata.

Response 2: The number of stomata was determined in the lower epidermis of the leaves and observations were made using a microscope at 100x, with 10 fields of view taken from each leaf for counting. In the reference I cited below, the method has a measurement for the number of stomata, using a field of view of 0.04 mm² for the measurement, whereas I used the entire field of view, repeated 10 times. (In 2.4.4. Highlighted in yellow)

（Reference: Zhou, Y., Jiang, X., Schaub, M. et al. Ten-year exposure to elevated CO2 increases stomatal number of Pinus koraiensis and P. sylvestriformis needles. Eur J Forest Res 132, 899–908 (2013). https://doi.org/10.1007/s10342-013-0728-8 ）

Comments 3: Missing data on confirmation of the ploidy of the original material.

Response 3: The original Begonia×benariensis material was purchased from Benary in Germany and is of the F1 generation, with high stability. Begonia×benariensis belonging to the genus Begonia L. in the family Begoniaceae C. Agardh.

Plants under the genus Begonia L. are all diploid, but chromosome numbers may vary from one variety to another. This has been demonstrated in many references, e.g: Oginuma, K., Peng, CI. Karyomorphology of Taiwanese Begonia (Begoniaceae): taxonomic implications. J Plant Res 115, 0225–0235 (2002). https://doi.org/10.1007/s102650200028, Yoshiko Kono, Ching-I Peng, Kazuo Oginuma , Hsun-An Yang, Yan Liu, Kuo-Fang Chung. Cytological Study of Begonia Sect. Diploclinium (Begoniaceae). CYTOLOGIA 86, 359-366 (2021). https://doi.org/10.1508/cytologia.86.359.

Chromosome numbers have also been added to the abstract section.

Comments 4: Unknown software for ploidy analysis.

Response 4: Flow cytometry software for ploidy analysis has been supplemented. On page 4, line 139.

Comments 5: According to the mentioned references, it is problematic to track down and verify the cited sources. In addition, many regional resources are not published in English, except with a very brief abstract.

Response 5: All the references mentioned can be found in the China National Knowledge Infrastructure, through which you can search all the information about this reference, including journals, fund sponsorships, categories etc. Each reference has a corresponding DOI number, and it can also be searched in Google Scholar. The China National Knowledge Infrastructure is the largest literature retrieval system in China, and scholars from all over the world can access the website to search for papers about China or abroad.

We have also replaced some Chinese references with English references.

4. Response to Comments on the Quality of English Language

Point 1:  Extensive editing of English language required

Response 1: MDPI English language editing services was used to enhance the English level.

5. Additional clarifications

Response : Some references of little relevance was removed.

  Point-by-point response to Comments and Suggestions for Authors

Comments 1: Line 29 Do not italicise the authors of the scientific names of plants.

Response 1: Italics have been removed from the author's name of the scientific name in line 28. Begonia in the front is also italicized

Comments 2: Lines 135 and 140 Supplement the manufacturer of the flow cytometry equipment used. Also indicate the license holder of the software used.

Response 2: Lines 134 and 140 add the manufacturer of the equipment and the license holder of the software. (Highlighted in yellow)

Comments 3: Table 2 It is necessary to explain the abbreviation “CK“ below the table.

Response 3: Explanations for CK are added below both Tables 2 and 3. (Highlighted in yellow)

Comments 4: Line 148 Specify the nylon mesh size in micrometers.

Response 4: Changed the nylon mesh unit to micron in line 148.

Comments 5: Line 352 Paragraph 4.2. Write the name of the genus Begonia in italics.

Response 5: Paragraph 4.2 "Begonia" in the title has been italicised.

Re-check the whole text and change the un-italicised Begonia to italics in 113, 124, 169, 205, 224, 281.

Also unabbreviate the B. in the titles of 3.3.2 and 3.3.3.

Comments 6: Although authors  explain how stomata were measured, standard methods recommend evaluating stomata abundance per unit area expressed in micrometers or square milimeters. Can you convert the area unit?

Response 6: The original number of stomata per unit area has been changed to stomatal density and expressed (No/mm²) as unit.

Replace all numbers of stomata per unit area in the manuscript with stomatal density.(Lines 17、19、166、263、272、275、395)

Comments 7: Available DOI addresses should be listed in references. It would be allow for better resource searches.

Response 7: DOI numbers have been added to all references.

Since the references corresponding to 5, 25, and 30 do not have DOI numbers due to being too old. So we replaced the fifth reference with a new one, replaced the 25th with a new one while the corresponding text was also modified (In page 11, lines 361-363), and deleted the 30th.

4. Response to Comments on the Quality of English Language

Point 1:  Minor editing of English language required

Response 1: MDPI English language editing services was used.

5. Additional clarifications

No