Physio-Anatomical Study of Polyploid Watermelon Grafted by Different Methods

Round 1

Reviewer 1 Report

After carefully reviewing the manuscript I concluded that the authors sufficiently addressed my comments.

I suggest that the article is accepted for publication in Agronomy.

Thank you for giving me the opportunity to assess the manuscript.

Author Response

We thank the very professional, very dedicated editor and reviewers of our submitted manuscript.

Please see the manuscribt

Reviewer 2 Report

This manuscript written by Kaseb et al. provides study on grafting of polyploid watermelon. The authors performed grafting using diploid, triploid and tetraploid watermelon and addressed the effect of ploidy to grafting characters. Physiological characterization described the difference of each graft combination. The detailed descriptions presented in this study are worth to be shared in this field. I provide minor points.

L112. watermelon -> squash?

L94-95. Need to be corrected.

L119. tell 12 days -> till 12 days after grafting?

L127. Three grafting methods were used for Diploid, Triploid, and Tetraploid watermelon grafting -> Three grafting methods were used for watermelon grafting

L127-128. Legend of Figure 1 does not enough explain the figure panels.

L132-133. I cannot read.

L152. Provide provider information for the SOD assay kit/ YX-C-A500. Same for other kits and equipment.

L172. “Di, Tri, and Tetra watermelon” I understand but need to be described correctly.

L194. I don’t clearly understand the meaning of “among diploid (Di), 194 triploid (Tri), and tetraploid (Tetra)”. Does it mean between Di and Di, Tri and Tri, and Tetra and Tetra? Or all possible combinations were tested? Please clarify. Maybe this part is explained in L132-133.

Author Response

• L112. watermelon -> squash?

We are using Diploid, Triploid, and Tetraploid watermelon as scion

and squash interspecific hybrid between (Cucurbita moschata × Cucurbita maxima) as rootstock

Response: We changed the sentences as follow:

Polyploid seeds for one variety (mimei), which is homozygous and genetically stable, and passed the achievement appraisal of the Chinese Department of Agriculture in 1990 and won the second prize of science technology progress of the Department of Agriculture in 1991 [54], were used as scions and squash as rootstock which is an interspecific hybrid between (Cucurbita moschata × Cucurbita maxima) (xijiaqiangsheng) and widely used in China as a rootstock, obtained from the polyploid watermelon group - Zhengzhou Fruit research institute (CAAS) China.

L94-95. Need to be corrected.

Response: We changed the sentences as follow:

Oda suggested that the reduction in the difference in diameter of the hypocotyls between the scion and rootstock was effective in increasing the survival rate and promoting the growth of cucumber plants, while the number of vascular bundles did not affect the survival rate.

• L119. tell 12 days -> till 12 days after grafting?

Yes, after grafting

Response: We added (after grafting)

• L127. Three grafting methods were used for Diploid, Triploid, and Tetraploid watermelon grafting -> Three grafting methods were used for watermelon grafting

Response: We rewrite this sentence (Three grafting methods were used for watermelon grafting)

• L127-128. Legend of Figure 1 does not enough explain the figure panels.

Response: We rearrange the figure and add the explanation

Figure 1. Three grafting methods were used for watermelon grafting, (A) Tongue grafting method (T), (B) Splice grafting method (S), and (C) Hole grafting method (H).

• L132-133. I cannot read.

Response: We rewrite this paragraph

Survive rates were measured after 15 days from the grafting by counting the successful seedlings and dividing them by the total number of the grafting seedlings. The individual plants were counted from three biological replicates; each replicate contains 30 plants from the splice and hole grafting methods (which had significant differences between polyploids).

• Provide provider information for the SOD assay kit/ YX-C-A500. Same for other kits and equipment.

Response: We rewrite this paragraph

Superoxide dismutase (SOD) activity in the supernatant was assayed for its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) by reading the absorbance at 560 nm wavelength using SOD assay kit/ YX-C-A500 for the measurement of SOD activity. Catalase activity was determined by measuring the rate of change of H2O2 absorbance in 60 s at 240 nm using a spectrophotometer by CAT assay kit/BC0200.

Peroxidase (POD) can catalyze the oxidation of phenols and amines by H2O2, which has the dual function of eliminating the toxicity of H2O2, phenols and amines. POD catalyzes the oxidation of specific substrates by H2O2, and has characteristic light absorption at 470nm using POD assay kit/ YX-C-A502. H2O2 forms a yellow titanium peroxide complex with titanium sulfate, with characteristic absorption at 415 nm wavelength using H2O2 assay kit/ YX-C-A400 (Sino best biological technology co, Beijing, China) according to the manufacturer’s instructions. Three biological replications from grafting union at 0, 3, and 15 DAG from three different plants for each replicate were collected for analysis.

• L172. “Di, Tri, and Tetra watermelon” I understand but need to be described correctly.

Response: We rewrite this paragraph

Five samples (graft union region) were collected from each replicate in Diploid, Triploid, and Tetraploid watermelons after 15DAG, which grafted with splice and hole grafting method

• L194. I don’t clearly understand the meaning of “among diploid (Di), 194 triploid (Tri), and tetraploid (Tetra)”. Does it mean between Di and Di, Tri and Tri, and Tetra and Tetra? Or all possible combinations were tested? Please clarify. Maybe this part is explained in L132-133.

Response: We rewrite this paragraph

Plant Survive rate was measured at 15DAG in diploid (Di), triploid (Tri), and tetraploid (Tetra) watermelons which were grafted by using Tongue (T), Splice (S), and Hole (H) grafting method during two consecutive seasons, March and August 2019.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The authors studied the impact of diploid, triploid and tetraploid watermelon scions grafted onto an interspecific squash hybrid, as well as three (results from only two are presented) grafting techniques on the antioxidant and sugar contents, hormonal and anatomical profiles, and root development of the derived grafted seedlings.

The introduction provided sufficient information about the scope of the research (as stated mainly by the abstract). However, the objectives must be thoroughly revised since they are not clear.

I did not quite understand why the authors did not present more results about the Tongue grafting method. A few more details about the experimental procedures should be included in the Materials and methods.

The discussion is very weak and should thoroughly be revised. Many of the results are merely compared with literature findings while almost no explanations and theories are provided regarding the rather important data. Moreover, some results (i.e. root growth and development, and anatomical observations) are not even referred to in the discussion.

To further improve the text, I suggest the following changes in the manuscript.

L11. I would say that watermelon “is one of the most desirable vegetable crop…”

L24. Keywords must not be included in the title. Please remove or substitute “polyploid watermelon”. Moreover, “mechanism” is too vague and general.

L49-50. Please rephrase the sentence in order to be clearer.

L89-91. I suggest that you thoroughly revise the objective paragraph since the aim of the study is completely unclear.

L97-98. Please state the species of the interspecific squash hybrid.

L99-103. Could you please explain why you did not show more results about the Tongue method?

L103-104. Please, also include the period the experiment was conducted (such as in L176-177), as well as information about the substrates, light conditions (supplementary lighting?), and other details during the seedling growth.

L110-114. This has already been mentioned in the introduction. Please remove this part.

L124-125. Did you use artificial lighting in the healing room? How many days did the seedlings stay in the healing room?

L129-130. Did you perform separate analysis for the scion and rootstock?

L178-179, 181, 183. Please do not mention in the text values already visible in the figures. Alternatively, you may mention differences among them.

L186 Figure 2. When showing percentages, y axis should always have values 0-100%

L369. Figure 9 should be moved after the first time it is mentioned in the text (i.e. after the next paragraph).

L386. Letters in Figure 10 are very small and it is difficult to read and observe.

L390. You did not discuss root growth and development, as well as anatomical observations. These results seem important but you did not include any explanations or similar literature findings.

L391-406. Most information in these paragraphs is already mentioned in the introduction. Please remove/alter this content and avoid similar repetitions.

L407-420. Most of this paragraph is a repetition from the results section. Please include possible explanations about why the hormones studied fluctuated depending on the –ploid state and grafting technique.

L421-440. In this paragraph you compare (and reaffirm) your results with other studies. I would also expect some kind of explanation or theory about why your experiments led to these results.

L441-459. Similarly to the above paragraphs, you should strengthen your discussion by providing possible explanations and theories about your findings.

L466-473. The conclusions should include take-home messages about your main findings. In essence, what should the reader remember from this manuscript. The results of this study are quite important but are not adequately presented and discussed.

Author Response

Comments and Suggestions for Authors

The authors studied the impact of diploid, triploid and tetraploid watermelon scions grafted onto an interspecific squash hybrid, as well as three (results from only two are presented) grafting techniques on the antioxidant and sugar contents, hormonal and anatomical profiles, and root development of the derived grafted seedlings.

The introduction provided sufficient information about the scope of the research (as stated mainly by the abstract). However, the objectives must be thoroughly revised since they are not clear.

I did not quite understand why the authors did not present more results about the Tongue grafting method. A few more details about the experimental procedures should be included in the Materials and methods.

The discussion is very weak and should thoroughly be revised. Many of the results are merely compared with literature findings while almost no explanations and theories are provided regarding the rather important data. Moreover, some results (i.e. root growth and development, and anatomical observations) are not even referred to in the discussion.

To further improve the text, I suggest the following changes in the manuscript.

Dear Editors and Reviewers,

We thank the very professional, very dedicated editor and reviewers of our submitted manuscript. We have incorporated the suggested changes, and a response to each of the reviewers' comments is added below as we try to address their concerns.

1. L11. I would say that watermelon “is one of the most desirable vegetable crop…”

Response: The changes have been made accordingly and highlighted

Watermelon is one of the most desirable vegetable crops in the world

2. L24. Keywords must not be included in the title. Please remove or substitute “polyploid watermelon”. Moreover, “mechanism” is too vague and general.

Response: The changes have been made accordingly and highlighted

We removed these keywords and added Genome duplication

3. L49-50. Please rephrase the sentence in order to be clearer.

Response: The changes have been made accordingly and highlighted

We removed this sentence

4. L89-91. I suggest that you thoroughly revise the objective paragraph since the aim of the study is completely unclear.

Response: The changes have been made accordingly and highlighted

This research studied the effect of genome doubling on graft compatibility at physiological and anatomical levels, which may be led to a good explanation of the compatibility mechanisms. It will also open the door for breeders to produce tetraploid rootstocks, which have high survival rates and high resistance to biotic and abiotic stress.

5. L97-98. Please state the species of the interspecific squash hybrid.

Response: The changes have been made accordingly and highlighted

And squash interspecific hybrid (Cucurbita moschata × Cucurbita maxima) (Xijiaqiangsheng)

6. L99-103. Could you please explain why you did not show more results about the Tongue method?

We are working in polyploid breeding and production. Our main work in this study is grafting to study the compatibility mechanisms and the effect of genome doubling in graft compatibility. We started this study with a wide idea using three grafting methods and three polyploids. From the grafting methods, we choose the two methods which have high significant differences between polyploids. And the second step in the future, we choose the Splice method, which has high significant differences between polyploids to study RNA-seq.

7. L103-104. Please, also include the period the experiment was conducted (such as in L176-177), as well as information about the substrates, light conditions (supplementary lighting?), and other details during the seedling growth.

Response: The changes have been made accordingly and highlighted lines (102-105)

Plants were cultivated in a glass-house with the growth conditions were set to 25-30℃ with 60-85% humidity with ordinary light. The sensor in each experimental plot's center was used to record temperature and humidity (THtool-V151_En; Campbell Scientific Ltd., China).

8. L110-114. This has already been mentioned in the introduction. Please remove this part.

Response: The changes have been made accordingly and highlighted

We removed this part

9. L124-125. Did you use artificial lighting in the healing room? No

How many days did the seedlings stay in the healing room?

We closed the healing room for seven days. After that, we start to open gradually tell 12 days, and after 15 days, we collected survival rates data and samples.

10. L129-130. Did you perform a separate analysis for the scion and rootstock? No

This study aims to compare polyploid watermelons using different grafting methods, which have significant differences in the survival rates, to study the compatibility mechanisms, leading to understanding this mechanism. Also, to study the effect of genome doubling on graft compatibility.

L178-179, 181, 183. Please do not mention in the text values already visible in the figures. Alternatively, you may mention differences among them.

Response: The changes have been made accordingly and highlighted

The results showed that the splice method was more compatible with tetraploid and triploid and gave a higher success rate than the diploid. On the contrary, the hole method was compatible with diploid more and gave a survival rate higher than the tetraploid. While the tongue method was lower in survival rate results and no high differences between polyploids, the following measurements will be completed on the splice and hole methods to study compatibility mechanisms and study the effect of genome duplication in graft compatibility.

11. L186 Figure 2. When showing percentages, y axis should always have values 0-100%

Response: The changes have been made accordingly

12. L369. Figure 9 should be moved after the first time it is mentioned in the text (i.e. after the next paragraph).

Response: The changes have been made accordingly and highlighted

13. Letters in Figure 10 are very small and it is difficult to read and observe.
14. L390. You did not discuss root growth and development, as well as anatomical observations. These results seem important but you did not include any explanations or similar literature findings.

Response: The changes have been made accordingly and highlighted

We added part in the introduction

Rootstock-scion compatibility depends on anatomical, physiological, and genetic variables. Oda et al. (1993) [51] Suggested that more minor differences between the cucumber scion and the squash rootstock in their hypocotyls' diameters might increase compatibility. Still, the numbers of vascular bundles did not affect compatibility. The greater the differences between the rootstock and the scion, the lower the success rate. Traka-Mavrona et al. (2000) [52] also showed that the differences in stem diameter between Cucurbita and Cucumis reduced grafts' survival rate.

And added this part in the discussion

Rootstock-scion compatibility depends on anatomical, physiological, and genetic variables and the survival rate increases with fewer differences between origin and taste [51]. The survival rate was inversely related to the difference between the diameters of the scion and the rootstock. Our anatomical study showed that the adhesion region in tetraploid with the splice method was long compared to the hole method. Since the tetraploid seedling is flat (not round), the hole method is not suitable for grafting. In the diploid, the slides showed that the adhesion area in the hole is bigger and longer than the splice; this may explain the high survival rate in the diploid with the hole method. Likewise, for triploid, the splice's adhesion area was bigger than the hole, explaining the increased survival rate in the splice more than the hole.

L391-406. Most information in these paragraphs is already mentioned in the introduction. Please remove/alter this content and avoid similar repetitions.

Response: The changes have been made accordingly and highlighted

We removed this part

L407-420. Most of this paragraph is a repetition from the results section. Please include possible explanations about why the hormones studied fluctuated depending on the –ploid state and grafting technique.

Response: The changes have been made accordingly and highlighted

We rewrite this part

In this study, we compared hole, splice, and tongue grafting methods among diploid, triploid, and tetraploid watermelon. The data showed that the hole method gave a high survival rate in Di and Triploid watermelon, while the splice method gave a high survival rate with tetraploid watermelon. While with the tongue method, the differences were not very high.

It has been reported that plant hormones play a vital role in callus initiation and vascular bundle formation at grafting union [30-32,35,36,59-66]. During the grafting process, the maximum contents of IAA were observed till 3 DAG in the scion [66,67]. We compared the IAA and ZR contents in Di, Tri, and Tetra watermelon graft unions (Figure 3) using splice and hole grafting methods on different days after grafting. The IAA and ZR contents in Tetra combinations were significantly high compared to Di and Tri combinations with the splice method. Di and Tri had a high content of IAA and ZR when the hole method was used.

Our results showed high compatibility in Tetra grafted with splice method and high content and high increment rates of hormones than Di, especially at 3 DAG (critical period) and 15 DAG. While Di and Tri gave high survival rate with hole method and high increment rates of IAA and ZR than Tetra, these results can explain high survival rates in Tetra with the splice and high survival rate in Di and Tri with hole method, these results were in agreement with [66,70,71].

L421-440. In this paragraph you compare (and reaffirm) your results with other studies. I would also expect some kind of explanation or theory about why your experiments led to these results. Response: The changes have been made accordingly and highlighted

We rewrite this part

The grafting process faced more stresses like injury or wound, complete dark, and high humidity during healing time [61,80]. The most critical time in the healing process is the 2nd and 3rd days after grafting. [66]. Polyploids are more tolerant of abiotic stresses than Di, and genome doubling gave high resistance to stresses because of the high contents of hormones and antioxidants' high activities [55,72-74].

The results in both seasons showed that POD, SOD, and CAT activities were significantly different between different ploidy watermelons among splice and hole methods (Figure 4).  In this study, POD, SOD, and CAT activities showed a high increase during the Tetra healing process with the splice method, while the increment rates were high in Di and Tri with hole method. On the contrary, the content of hydrogen peroxide (H2O2) didn't increase during the healing process in Tetra with the splice method. It didn't increase in Di and Tri with the hole method. These results were because of the high activities of antioxidants, which leads to scavenging oxygen radicals and H2O2. According to [43,46].

15. L441-459. Similarly, to the above paragraphs, you should strengthen your discussion by providing possible explanations and theories about your findings.

Response: The changes have been made accordingly and highlighted

We rewrite this part

Carbohydrates provide energy to plants, and the cotyledons are the source of carbohydrates in the seedlings [27,50]. The survival rate was positively correlated with an increase in starch content [51]. An increase in the percentage of carbohydrates before grafting by adding sucrose to the rootstocks led to a higher survival rate [50]. In our results, with the splice method, the accumulation of sugars and starch contents in the graft union at 0DAG was significantly higher in Tetra than in Tri and Di. The decrement rates of sugars and starch were meager compared to Di and Tri. These results may explain the increment in the survival rate in Tetra with the splice method. Same with the hole method, Di and Tri gave a higher survival rate because of the higher content of starch and sucrose at 0DAG and the lower decrement rates during 3DAG than tetraploid watermelon. These results are consistent with [81], who suggested that higher carbohydrate contents in grafted tissues could lead to successful grafting.

Just as hormones are essential in the process of compatibility and callus formation, so are carbohydrates [50,51,81-83]. The experiment has proved that increasing the percentage of carbohydrates increased the grafting survival rate [50].

16. L466-473. The conclusions should include take-home messages about your main findings. In essence, what should the reader remember from this manuscript. The results of this study are quite important but are not adequately presented and discussed.

This study investigated physiological and anatomical changes in graft-compatible and graft-incompatible polyploid watermelon with different grafting methods. Our results suggest that the higher content of hormones, antioxidants activities, and carbohydrates, would contribute to superior physiology, callus initiation, differentiation, and growth performance in the graft-compatible combination. These results provide us some advice such as exogenous applications with sugars or hormones before grafting and reduce the stresses on the plant during the healing period to increase the survival rate. Also, to study the effect of genome doubling on graft compatibility. Our findings should further understand the compatibility mechanisms to improve plant growth performance and cause incompatible-rootstock grafting to influence plant growth. Also, it can open the door for breeders to produce tetraploid rootstock, which has high compatibility and high resistance for abiotic and biotic stresses.

Finally, we are incredibly thankful to both the reviewers for their valuable time. We have incorporated all the changes suggested by both reviewers. Moreover, we have thoroughly revised the entire text to remove existing grammatical and syntax errors to improve writing quality and clarity. We hope that our revised paper fulfills the rigor required by the journal for publication. We will be happy to hear any further suggestions from the reviewers.

Sincerely,

Mohamed Omar Kaseb, Muhammad Jawad Umer, and Wenge Liu, on behalf of all authors

Reviewer 2 Report

Dear authors

This research can provide beneficial information for the vegetable industry. I would suggest avoiding to generalize too much different results. The authors only used one scion and one rootstock comparison so it is difficult to obtain a general all encompassing conclusion from this work. All the statistical analyses need to be rechecked. The authors have compared all the treatments combinations all together without describing if the effects from the different factors are significant or not. The same is the case with the comparisons across seasons. Similarly, ANOVA is being used and I believe the assumptions for the correct use of ANOVA have not been checked. The authors need to add the number of plants that they grafted per treatment to obtain the survival rates. 

I decided to stop reviewing the document after the second set of results because all the write-ups are based on comparisons across all treatments. If the correct statistical analyses was performed, then some of those results could be presented in a different manner that can be easy to understand to the reader.

Comments for author File: Comments.pdf

Author Response

Look at pdf file

Dear Editors and Reviewers,

We thank the very professional, very dedicated editor and reviewers of our submitted manuscript. We have incorporated the suggested changes, and a response to each of the reviewers' comments is added below as we try to address their concerns.

This research can provide beneficial information for the vegetable industry. I would suggest avoiding to generalize too much different results. The authors only used one scion and one rootstock comparison so it is difficult to obtain a general all-encompassing conclusion from this work. All the statistical analyses need to be rechecked. The authors have compared all the treatments combinations all together without describing if the effects from the different factors are significant or not. The same is the case with the comparisons across seasons. Similarly, ANOVA is being used and I believe the assumptions for the correct use of ANOVA have not been checked. The authors need to add the number of plants that they grafted per treatment to obtain the survival rates. 

I decided to stop reviewing the document after the second set of results because all the write-ups are based on comparisons across all treatments. If the correct statistical analyses was performed, then some of those results could be presented in a different manner that can be easy to understand to the reader.

Response: We changed the analysis as follow:

The present experiment was designed based on a completely randomized design. The general linear model by two-way ANOVA, 2 × 3 were applied to analyze the data using SPSS 26 (IBM).

The two-way ANOVA model included the main effects of species (diploid or triploid or tetraploid), the grafting method (Splice or Hole), and interactions between species and grafting methods.

2- Not always true. I have screening colchicine-induced tetraploid from diploid plants that don't have any advantage in comparison with their diploid counterpart.

Response: The success rate of dealing with colchicine does not exceed 0.05%. However, when the duplication in watermelons is successful, there are apparent differences between the diploid and the tetraploid. It can be distinguished outwardly easily by the female flower, where it is significantly large compared to the diploid and the thickness and size of the leaf.

Fig. 2. Morphological features of diploid (CLD1) and tetraploid (CLT1) plants. Detailed morphological features of diploid and tetraploid Charleston Gray field-grown watermelon. (a) Phenotype of field-grown plants 70 d after planting. (b) Morphology of leaves detached from mother plants. (c) Comparison of stems collected from the middle of the main stem showing variation in width and trichome morphology. (d) Phenotypic variation of staminate flowers collected during flowering. (e) Fruits showing no significant morphological difference between diploid and tetraploid. Bars, 5cm (b, e); 1cm (c, d) (Saminathan et al., 2015).

Figure 5.4 A comparison between the leaf and flower of a (A) diploid and (B) induced tetraploid watermelon illustrating the gigas effect (http://plantbreeding.coe.uga.edu/index.php?title=5._Polyploidy)

3- Line 50: I believe you can add the name of the author in sentences like this

Response: We added the author’s name

3- Line 68: This is probably only one reason. There are anatomical and mechanical reasons as well. There is too much generalization in the text.

Response: We rewrite this part

3- Line 106: This paragraph needs to be re-written. It doesn't read well. Also, did you evaluate seedlings only for a total length of 15 days? What is the cycle of growth and production of a watermelon plant? In fruit trees, we can see something called delayed incompatibility. Also, incompatibility can be expressed as well by reduction of fruit production. In addition, there are other physiological responses.

Response: We added this part line 101-110

Plants were cultivated in a glass-house with the growth conditions were set to 25-30℃ with 60-85% humidity with ordinary light. The sensor in each experimental plot's center was used to record temperature and humidity (THtool-V151_En; Campbell Scientific Ltd., China). We closed the healing room for seven days. After that, we start to open gradually tell 12 days, and after 15 days, we collected survival rates data and samples.

3- Line 108: We need information of how many plants were grafted and how many units per different treatment.

Response: We added this part line 128-137

Samples (grafting union) were taken as three biological replicates. Every replicate of 30 plants from the splice and hole methods (which had highly significant differences between polyploids) was collected at three different stages 0, 3, and 15 days after grafting for the determinations and histological studies. Samples were taken at 0DAG as a control to study the grafting process development. At 3DAG is the critical time for grafting, as it has the highest rate of cellular activity, which is determined based on compatibility/incompatibility in the grafting. At 15DAG, samples were taken after survival and success compatibility. The most critical time in the healing process is 2-3 DAG during the healing stage (Gainza, Opazo, & Muñoz, 2015; Schwarz, Rouphael, Colla, & Venema, 2010), there is the highest rate of stress (complete dark, high humidity, and temperature) and the highest increase of hormones and antioxidants leads to graft compatibility (Melnyk, Schuster, Leyser, & Meyerowitz, 2015).

3- Line 163: Did survival rate met test for normality? In most of the cases this is not true. I believe you should check first the assumptions for ANOVA before running the tests. You may need to transform the survival data before doing the analyses.

Response: We changed the analysis

Finally, we are incredibly thankful to both the reviewers for their valuable time. We have incorporated all the changes suggested by both reviewers. Moreover, we have thoroughly revised the entire text to remove existing grammatical and syntax errors to improve writing quality and clarity. We hope that our revised paper fulfills the rigor required by the journal for publication. We will be happy to hear any further suggestions from the reviewers.

Sincerely,

Mohamed Omar Kaseb, Muhammad Jawad Umer, and Wenge Liu, on behalf of all authors

Gainza, F., Opazo, I., & Muñoz, C. (2015). Graft incompatibility in plants: Metabolic changes during formation and establishment of the rootstock/scion union with emphasis on Prunus species. Chilean Journal of Agricultural Research, 75, 28-34. doi:10.4067/S0718-58392015000300004

Melnyk, C. W., Schuster, C., Leyser, O., & Meyerowitz, E. M. (2015). A Developmental Framework for Graft Formation and Vascular Reconnection in Arabidopsis thaliana. Curr Biol, 25(10), 1306-1318. doi:10.1016/j.cub.2015.03.032

Saminathan, T., Nimmakayala, P., Manohar, S., Malkaram, S., Almeida, A., Cantrell, R., . . . Vajja, V. G. (2015). Differential gene expression and alternative splicing between diploid and tetraploid watermelon. Journal of Experimental Botany, 66(5), 1369-1385.

Schwarz, D., Rouphael, Y., Colla, G., & Venema, J. H. (2010). Grafting as a tool to improve tolerance of vegetables to abiotic stresses: Thermal stress, water stress and organic pollutants. Scientia Horticulturae, 127(2), 162-171. doi:10.1016/j.scienta.2010.09.016

Saminathan, T., Nimmakayala, P., Manohar, S., Malkaram, S., Almeida, A., Cantrell, R., . . . Vajja, V. G. (2015). Differential gene expression and alternative splicing between diploid and tetraploid watermelon. Journal of Experimental Botany, 66(5), 1369-1385.