Propagation of Hinoki Cypress (Chamaecyparis obtusa) Through Tissue Culture Technique as a Sustainable Method for Mass Cloning of Selected Trees

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper presents a novel micropropagation protocol for a species of cypress native to Japan. The authors successfully sterilized the explants (leaf segments) derived from adult trees, enhanced multiple bud formation and shoot elongation through several consequent experiments followed by rooting and successful acclimatization, thus concluding the entire micropropagation cycle. Although there were earlier attempts to micropropagate this species, the data presented in this study are novel and will be of particular interest to horticulturists, plant tissue culture specialists, and tree physiologists worldwide. The paper is generally well-written and may be accepted after minor revision, as recommended below.

 

1. In the abstract, I recommend including data on acclimatization because this is an essential step towards mass production and is often difficult to achieve for micropropagated trees.

 

2. Materials and methods, 2.1. Please indicate how many trees were collected for each clone.

 

3. In the same paragraph, please indicate how long it took for cuttings to form roots and the conditions in a greenhouse.

 

4. Under 2.2, please indicate the diameter of Petri plates used for adventitious bud induction. Under 2.4 please add the volume of bio-flasks for shoot cultivation.

 

5. For each experiment, please mention the timepoint for data collection (this is only given in the results, but would be helpful in Materials and Methods as well).

 

6. It seems that the range of responses to different treatments vary considerably between clones. Therefore, it would be interesting to see the data regarding each clone response to different PGR in the media for adventitious bud induction (Fig. 1), shoot induction (Fig. 3) and rooting (Fig. 4). Consider including those data for each clone as a Supplementary.

 

7. The last paragraph in the Discussion describing acclimatization may be moved to the Results section because it is an important achievement that is worth to be mentioned.

 

In summary, I would like to congratulate the authors for their accomplishment in conducting interesting and successful research on a subject that posed significant challenges.

Author Response

Response to Sustainability Reviewer １

Dear Reviewer １,

We would like to thank you for the constructive comments to improve the quality of our manuscript. We have revised the manuscript accordingly with your comment as described below (modifications in the Revised version have been highlighted in red and yellow).

 

Best regards,

 

Tsuyoshi E. Maruyama

Corresponding Author

14 March 2025

 

 

Comments and Suggestions for Authors

This paper presents a novel micropropagation protocol for a species of cypress native to Japan. The authors successfully sterilized the explants (leaf segments) derived from adult trees, enhanced multiple bud formation and shoot elongation through several consequent experiments followed by rooting and successful acclimatization, thus concluding the entire micropropagation cycle. Although there were earlier attempts to micropropagate this species, the data presented in this study are novel and will be of particular interest to horticulturists, plant tissue culture specialists, and tree physiologists worldwide. The paper is generally well-written and may be accepted after minor revision, as recommended below.

1. In the abstract, I recommend including data on acclimatization because this is an essential step towards mass production and is often difficult to achieve for micropropagated trees.

*According your suggestion, we are including data of acclimatization in Abstract.

 

2. Materials and methods, 2.1. Please indicate how many trees were collected for each clone.

* According your suggestion, we are including information of tree collected for each clone in Material and methods..

 

3. In the same paragraph, please indicate how long it took for cuttings to form roots and the conditions in a greenhouse.

* According your suggestion, we are including information of cuttings and conditions in greenhouse.

 

4. Under 2.2, please indicate the diameter of Petri plates used for adventitious bud induction. Under 2.4 please add the volume of bio-flasks for shoot cultivation.

* According your suggestion, we are including information of diameter of Petri plates and volume of bio-flasks.

 

5. For each experiment, please mention the time point for data collection (this is only given in the results, but would be helpful in Materials and Methods as well).

* According your suggestion, we are including information for data collection in Material and methods.

 

6. It seems that the range of responses to different treatments vary considerably between clones. Therefore, it would be interesting to see the data regarding each clone response to different PGR in the media for adventitious bud induction (Fig. 1), shoot induction (Fig. 3) and rooting (Fig. 4). Consider including those data for each clone as a Supplementary.

* I completely agree with your comment that the range of responses to different treatments varies considerably between clones. However, in our study, we first tested one clone to determine the best treatment and then compared the clones testing the optimal treatment previously obtained. In each case, we are including the name of the clone used to determine the optimal treatment. The useful suggestion of a clone difference in response to each PGR is interesting, but would require an enormous amount of experimentation to analyze. Even if it could be evaluated, it would be unrealistic to develop a laboratory protocol that would make a different optimal medium for each clone each time.

 

7. The last paragraph in the Discussion describing acclimatization may be moved to the Results section because it is an important achievement that is worth to be mentioned.

*According with your recommendation, we are moving part of the last paragraph in the Discussion describing acclimatization to the Result section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. The Abstract contains well-known elements of in vitro plant propagation methodology, but does not describe the optimal conditions found by the authors for hinoki cypress. Please also consider including in the Abstract the information obtained on clone-specific characteristics (‘clones related to the cultivar “Nangouhi” (Na18, Na14 x Isa, Na14-14, Isa x Na14, and NaS) were easier to root than those derived from the cultivar “ Shizuoka-KenZairai ” (SKZ5 and SKZ8)’).

 

2. Plant materials. From the text of this section, it seems that clones are plants derived from cuttings from a single specimen, which is clearly not the case. Please edit the text in the Methodology section to include a very brief description of what you understand clones to be and how they are remarkable.

 

     3. What was considered a replicate, how was it measured, and how many were there.

 

4. The appearance of roots during rooting is a quantitative process. What was counted as a rooted explant?

 

5. Which clone was used to test the efficacy of different variants of the PGRs? If different, there should be information on the reliability (or unreliability) of the clone*PGR interaction.

 

6. What was the continuity of media in the successive transfer of explants at different stages? There are 3 stages in the study with different sets of media. Thus, from each of the 6 media for bud induction it is possible to transfer explants to 3 media for shoot elongation and then to 4 media for rooting. A total of 6x3x4 options, each of which is unique.

 

7. Section 3.3. What are the peculiarities of shoot elongation in different clones?

 

8. In the legend to Fig. 2, please state that all clones were grown on the previously selected optimal medium containing 10 μM BAP and 1 μM 2,4-D, and in the legend to Fig. 5 that rooting of elongated shoots occurred on medium containing 1 μM IBA and 0.1 μM NAA. Figures should be self-contained and understandable independently of the rest of the text.

Author Response

Response to Sustainability Reviewer 2

 

Dear Reviewer 2,

 

We would like to thank you for the constructive comments to improve the quality of our manuscript. We have revised the manuscript accordingly with your comment as described below (modifications in the Revised version have been highlighted in red and yellow).

Best regards,

 

Tsuyoshi E. Maruyama

Corresponding Author

14 March 2025

 

 

Comments and Suggestions for Authors

1. The Abstract contains well-known elements of in vitro plant propagation methodology, but does not describe the optimal conditions found by the authors for hinoki cypress. Please also consider including in the Abstract the information obtained on clone-specific characteristics (‘clones related to the cultivar “Nangouhi” (Na18, Na14 x Isa, Na14-14, Isa x Na14, and NaS) were easier to root than those derived from the cultivar “ Shizuoka-KenZairai ” (SKZ5 and SKZ8)’).

* According with your suggestion, we modified the Abstract by highlighting the optimal conditions for each step including also the information obtained on clone specific characteristics among ‘Nangouhi’ and ‘ShizuokaKenZairai’ cultivars.

 

2. Plant materials. From the text of this section, it seems that clones are plants derived from cuttings from a single specimen, which is clearly not the case. Please edit the text in the Methodology section to include a very brief description of what you understand clones to be and how they are remarkable.

* The statement is correct. However, although the explants were not collected directly from the adult trees, this does not refute the cloning of the selected original genotypes. As is well known, explant rejuvenation via cuttings or grafting is a widely used technique for cloning adult trees, because micropropagation becoming even more difficult as the plants age. We edited the text describing in detail the origin of plant materials in Materials and Methods section.

3. What was considered a replicate, how was it measured, and how many were there.

* We have revised the Materials and Methods to clarify this, and added information on the replicates used for examinations.

 

4. The appearance of roots during rooting is a quantitative process. What was counted as a rooted explant?

* In our experiments we consider an explant rooted if it shows both emergence and subsequent growing roots of at less 10 mm.

 

5. Which clone was used to test the efficacy of different variants of the PGRs? If different, there should be information on the reliability (or unreliability) of the clone*PGR interaction.

* We consistently test the effects of PGRs on a specific clone (in each case, we are including in Material and Method section the name of the clone used to determine the best treatment) and then evaluate the performance of each clone under the conditions established there. The clone differences that we evaluated include differences in both clonal potential and responsiveness to the PGR treatment determined previously. The useful suggestion of a clone difference in response to each PGR is interesting, but would require an enormous amount of experimentation to analyze. Even if it could be evaluated, it would be unrealistic to develop a laboratory protocol that would make a different optimal medium for each clone each time.

 

6. What was the continuity of media in the successive transfer of explants at different stages? There are 3 stages in the study with different sets of media. Thus, from each of the 6 media for bud induction it is possible to transfer explants to 3 media for shoot elongation and then to 4 media for rooting. A total of 6x3x4 options, each of which is unique.

* As you mention, running a complete experiment with six media for bud multiplication, three media for shoot elongation, four media for rooting, and applying it to eleven clones represents a monumental amount of experimentation and analysis. As we explained in point 5, in our study, we first tested one clone to determine the optimal treatment and then compared the clones testing the optimal treatment previously obtained.

 

7. Section 3.3. What are the peculiarities of shoot elongation in different clones?

* In our study, we analyzed the effect of PGRs on shoot elongation using a single clone, applying the previously selected optimal treatment achieved in each clone to obtain shoot explants for rooting experiments. We did not observed morphological differences in shoot elongation among clones. However, the corresponding statistical comparisons were not performed. We have added information in Materials and Methods and Results sections to describe this.

 

8. In the legend to Fig. 2, please state that all clones were grown on the previously selected optimal medium containing 10 μM BAP and 1 μM 2,4-D, and in the legend to Fig. 5 that rooting of elongated shoots occurred on medium containing 1 μM IBA and 0.1 μM NAA. Figures should be self-contained and understandable independently of the rest of the text.

* According your suggestion we have added the information of medium in the legend of Fig. 2 and Fig. 5.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I think that this original method of the cypress cloning may be published in this journal. One of the interesting questions is related to natural septics or antioxidants. On the color figure we saw blue color, peculiar to not only Japan cypress, but peculiar to many Cypress species. Blue pigment appears to be azulene ( or plural azulenes) as shown recently for  some woody plants. These pigments were not seen on the earlier stages of the development of cypress, but here, in vitro culture, it has been occurred.  This finding may be of a special scientific  interest in Future.

Author Response

Response to Sustainability Reviewer 3

 

Dear Reviewer 3,

 

We would like to thank you for the constructive comments regarding to our manuscript. Our comment have been described below highlighted in red.

Best regards,

 

Tsuyoshi E. Maruyama

Corresponding Author

14 March 2025

 

 

Comments and Suggestions for Authors

I think that this original method of the cypress cloning may be published in this journal. One of the interesting questions is related to natural septics or antioxidants. On the color figure we saw blue color, peculiar to not only Japan cypress, but peculiar to many Cypress species. Blue pigment appears to be azulene (or plural azulenes) as shown recently for some woody plants. These pigments were not seen on the earlier stages of the development of cypress, but here, in vitro culture, it has been occurred. This finding may be of a special scientific interest in Future.

*We agree with your assessment that the topic of pigmentation in cypresses would be an interesting subject to study in the future. Thank you for your interesting comment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript developed in vitro propagation method by tissue culture for hinoki cypress clones. However, no replication was designed in the study, and the statistical analyses of data were not strong.

1. Title: the explants were not collected from adult trees, from cutting plants indeed.
2. M&M: Eleven clones were used for this study, and kinds of PGR treatments were designed. However, the details on experiment design is not clear in 2.2, 2.3, and 2.4, such as how many explants were used? How many replicates and how many explants each replicate?
3. Results: 3.1 and table 1, no replicate. How did the difference analysis among the clones?
4. Results: 3.2, 3.3, and 3.4, they are two-factor experiments (clones and PGRs), but the authors analyzed them seperately. We do not know the response of different clones to different PGRs and we also do not know interaction between clones and PGRs.

Author Response

Response to Sustainability Reviewer 4

 

Dear Reviewer 4,

 

We would like to thank you for the constructive comments to improve the quality of our manuscript. We have revised the manuscript accordingly with your comment as described below (modifications in the Revised version have been highlighted in red and yellow).

Best regards,

 

Tsuyoshi E. Maruyama

Corresponding Author

14 March 2025

 

 

Comments and Suggestions for Authors

This manuscript developed in vitro propagation method by tissue culture for hinoki cypress clones. However, no replication was designed in the study, and the statistical analyses of data were not strong.

1. Title: the explants were not collected from adult trees, from cutting plants indeed.

* The statement is correct. However, although the explants were not collected directly from adult trees, this does not refute the cloning of the selected original genotypes. As is well known, explant rejuvenation via cuttings or grafting is a widely used technique for cloning adult trees, because micropropagation becoming even more difficult as the plants age. We have described in detail the origin of plant materials in Materials and Methods section.

2. 
   M&M: Eleven clones were used for this study, and kinds of PGR treatments were designed. However, the details on experiment design is not clear in 2.2, 2.3, and 2.4, such as how many explants were used? How many replicates and how many explants each replicate?

* We consistently tested the effects of PGRs on a specific clone, then evaluated the performance of each clone under the conditions established there. We have revised the Materials and Methods to clarify this, and added information on the replicates used for examinations.

 

3. Results: 3.1 and table 1, no replicate. How did the difference analysis among the clones?

* The statistical analysis method was described in detail in Materials and Methods 2.6. As mentioned there, clonal differences in surface sterilization of adult leaf explants were evaluated by constructing generalized linear models (GLMs). Here, the objective variable is the sterilization status (1: aseptic or 0: non-aseptic), so the number of explants tested within each clone (60-124) is the number of replicates (total number of analyses N=1,074). Although the number of replicates is different for each clone, this GLM analysis can be used to show robust statistical results even when the number of replicates per treatment is different, or even for an objective variable such as germination rate whose error distribution does not follow a normal distribution. Therefore, it is difficult to present the number of replicates as an experimental design in the text, but we have included the total number of analyses of the model in Results 3.1., as follow: The GLM model (N=1,074) fit was better when clone was used as a variable, but there were no significant differences among clones (P > 0.05).

4. 
   Results: 3.2, 3.3, and 3.4, they are two-factor experiments (clones and PGRs), but the authors analyzed them seperately. We do not know the response of different clones to different PGRs and we also do not know interaction between clones and PGRs.

* The reviewer's comment that these are two-factor experiments with clones and PGRs is a misunderstanding. We consistently test the effects of PGRs on a specific clone and evaluate the performance of each clone under the conditions established there. The clone differences that we evaluated include differences in both clonal potential and responsiveness to the PGR treatment determined before.

* The useful suggestion of a clone difference in response to each PGR is interesting, but would require an enormous amount of experimentation to analyze. Even if it could be evaluated, it would be unrealistic to develop a laboratory protocol that would make a different optimal medium for each clone each time.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

In the current version, the clarity of the presentation has improved considerably. However, in my opinion, some further minor amendments are desirable. 

2.1 Plant materials and surface sterilisation of explants

I understand that the 11 clones used by the authors are most likely 11 genotypes obtained by crosses in the breeding process and then propagated vegetatively (clone varieties). However, there is another understanding of a clone. In this other understanding, 11 clones may represent 11 trees previously obtained by cuttings of the same genotype.  Consider clarifying, for example, ‘ All these clones (one tree per clone) represent different genotypes selected for their useful characteristics (shape, growth, wood quality) and used as breeding stock.’

Clarification of the purpose of cuttings for explant rejuvenation at micropropagation of adult trees would also be good to be inserted in this section of the Methodology.

 

2.2 Adventitious-bud induction

Please consider adding: Each treatment has 10 replicates (plates with one explant in each).

Please consider specifying a variant of the previous step medium in each of sections 2.3; 2.4 and 2.5. For example, all shoot elongation studies are performed using bud clusters previously obtained on medium supplemented with 10 μM BAP and 1 μM 2,4-D.

 

‘* In our experiments we consider an explant rooted if it shows both emergence and subsequent growing roots of at least 10 mm’. Please add this information to section 2.4.

Author Response

25 March 2025

 

Response to Sustainability Reviewer 2

 

Dear Reviewer 2,

 

Thank you very much for the valuable comments to improve the quality of our manuscript. We have revised the manuscript according to your comments and incorporated all the recommendations into the updated manuscript. (New modifications in the revised version have been highlighted in red and sky blue).

Best regards,

 

Tsuyoshi E. Maruyama

Corresponding Author

25 March 2025

 

 

Comments and Suggestions for Authors

In the current version, the clarity of the presentation has improved considerably. However, in my opinion, some further minor amendments are desirable.

2.1 Plant materials and surface sterilisation of explants

I understand that the 11 clones used by the authors are most likely 11 genotypes obtained by crosses in the breeding process and then propagated vegetatively (clone varieties). However, there is another understanding of a clone. In this other understanding, 11 clones may represent 11 trees previously obtained by cuttings of the same genotype. Consider clarifying, for example, all these clones (one tree per clone) represent different genotypes selected for their useful characteristics (shape, growth, wood quality) and used as breeding stock.

* As you recommended, the clarification was included in Materials and Methods (2.1).

 

Clarification of the purpose of cuttings for explant rejuvenation at micropropagation of adult trees would also be good to be inserted in this section of the Methodology.

* As you recommended, the purpose of cuttings was inserted in Materials and Methods (2.1).

 

2.2 Adventitious-bud induction

Please consider adding: Each treatment has 10 replicates (plates with one explant in each).

* As you recommended, the number of explants per plate was added in Materials and Methods (2.2).

 

Please consider specifying a variant of the previous step medium in each of sections 2.3; 2.4 and 2.5. For example, all shoot elongation studies are performed using bud clusters previously obtained on medium supplemented with 10 μM BAP and 1 μM 2,4-D.

* As you recommended, the previous step medium was included in sections 2.3 and 2.4 in Materials and Methods.

 

* In our experiments we consider an explant rooted if it shows both emergence and subsequent growing roots of at least 10 mm’. Please add this information to section 2.4.

* As you recommended, the clarification about ‘explant rooted’ was included in section 2.4 in Materials and Methods.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

1. The explants were not collected from adult trees. This title may lead to the ambiguity that the tissue culture technique was based on the explants collected from the selected adult trees directly.  Actually, they were from the cutting plants.
2. In 2.2, 2.3, and 2.4, why were different clones used as the materials? This gives the impression that the study is a patchwork.
3. I still think the data analysis based on Poisson distribution is not correct. For example, in 3.1, "there were no significant differences among clones (P > 0.05)". In table 1, the percentage of aseptic explants of clone NaS is 100%, but clone Isa is 72.5%. If the 72.5% data is from an average of three replicates (30 explants per replicate), I suspect that the difference between these two clones will be significant.

 

 

 

Author Response

25 March 2025

 

Response to Sustainability Reviewer 4

 

Dear Reviewer 4,

 

Thank you very much for the valuable comments to improve the quality of our manuscript. We have revised the manuscript according to your comments and have tried to address your concerns where possible, as described below. We thank you in advance for your understanding. The new changes in the revised version are highlighted in red and sky blue.

Best regards,

 

Tsuyoshi E. Maruyama

Corresponding Author

25 March 2025

 

 

Comments and Suggestions for Authors

1. The explants were not collected from adult trees. This title may lead to the ambiguity that the tissue culture technique was based on the explants collected from the selected adult trees directly. Actually, they were from the cutting plants.

 

* To avoid any ambiguity regarding the explant used, we have removed the term 'adult' from the title and from the legend of Figures. Similarly, to clarify the origin of the explants, we have described in all sections (Abstract, Materials and Methods, Results, Discussion and Conclusion) that the explants used were from plants propagated by cuttings derived from selected adult trees. In addition, we also insert in the top of Material and Methods section a clarification of the purpose of cuttings for explant rejuvenation to promote micropropagation of adult trees.

 

1. In 2.2, 2.3, and 2.4, why were different clones used as the materials? This gives the impression that the study is a patchwork.

 

* The answer to your question may not be convincing, nor do we want to argue about the ideal experimental design, but the reason we use different clones at each step is as follows. Our initial plan was to use a single clone to determine the optimal medium for each step of micropropagation. For the initial bud induction step, we selected one clone a priori to determine the effect of PGRs and then used the optimal medium to compare clones. However, for subsequent steps, due to the large heterogeneity of the experimental material obtained in the previous step with the selected clone, we decided to use the clone with the best response in terms of quantity and homogeneity as the experimental material. For example, to determine the optimal medium for shoot elongation, we selected the clone that developed the appropriate number of bud clusters with the most homogeneous diameter-size characteristics for the elongation treatments. Similarly, to determine the optimal medium for rooting, we selected the clone that developed the appropriate number of elongated shoots with the most homogeneous length-size characteristics for the rooting treatments. The plan to select a clone a priori for all steps of micropropagation was changed to selecting a clone by empirical observation for each step, with the aim of homogenizing the explant to reduce experimental error. We sincerely hope for your understanding.

 

1. I still think the data analysis based on Poisson distribution is not correct. For example, in 3.1, "there were no significant differences among clones (P > 0.05)". In table 1, the percentage of aseptic explants of clone NaS is 100%, but clone Isa is 72.5%. If the 72.5% data is from an average of three replicates (30 explants per replicate), I suspect that the difference between these two clones will be significant.

 

* The analysis we used makes ecological and biological sense and has become the mainstream analysis in recent years (Dobson and Barnett, 2018, https://doi.org/10.1201/9781315182780). Here, to follow the biological rule, Poisson distribution (which can be considered the same as the normal distribution if the number is large enough) and the exponential function, and the binomial distribution and logistic regression, are used for count data and percentage data modeling, respectively. The data in Table 1 (sterility rates) therefore used a binomial distribution for the error distribution. We have a total of 1074 replicates, the value with 1 aseptic or 0 non-aseptic for every explant. For this data, we are testing for differences between clones, as shown in the next figure. As a result, no coefficients significantly different from 0 (Pr(>|z|)) were estimated between clones.

 

 

GLM analysis code in R

# glm(formula = asept ~ clone, family = binomial, data = d.ase)

 

Coefficients:

              Estimate Std. Error z value Pr(>|z|)

(Intercept)  1.857e+01  7.910e+02   0.023    0.981

cloneFj-5   -1.648e+01  7.910e+02  -0.021    0.983

cloneFj-8   -1.615e+01  7.910e+02  -0.020    0.984

cloneI      -1.760e+01  7.910e+02  -0.022    0.982

cloneIxN14  -1.523e+01  7.910e+02  -0.019    0.985

cloneIz-3   -1.624e+01  7.910e+02  -0.021    0.984

cloneN14xI  -1.600e+01  7.910e+02  -0.020    0.984

cloneN18    -1.589e+01  7.910e+02  -0.020    0.984

cloneNO7-5  -1.769e+01  7.910e+02  -0.022    0.982

cloneOi-6   -1.420e+01  7.910e+02  -0.018    0.986

cloneS       5.199e-08  1.003e+03   0.000    1.000

 

We hope that the description we have given above helps you to understand our analysis.

Author Response File: Author Response.pdf

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

After revision, I agree to accept this manuscript for publication.