Influence of the Rootstock and the Ploidy Level of the Scion and the Rootstock on Sweet Orange (Citrus sinensis) Peel Essential Oil Yield, Composition and Aromatic Properties

Round 1

Reviewer 1 Report

This research compared the yield and composition of PEO among different rootstocks and different ploidy levels of scion-stock combinations with different methods and analyses. It is found that rootstock could affect both the yield and composition of PEO, and ploidy level of scion not rootstock could significantly affect the composition of PEO. However, two minor questions I would like to mention.

1. 28 and 25 compounds were detected in the two trials respectively. What caused the difference? The genotype of the scions? It should be mentioned in the discussion.
2.  Some minor expressions should be cautious. E.g, in 2.2.1 Raw materials, "Three lots of five fruits" and in 3.2 below Table 2, in the sentence 'On the other hand,......', '...of the essential oil. (Table 1 and Figure 4') should be changed into " ...of the essential oil (Table 2 and Figure 4)

Author Response

We thank the reviewer for the evaluation of our manuscript and for his suggestions. The answers to his questions are the following:

1. 28 and 25 compounds were detected in the two trials respectively. What caused the difference? The genotype of the scions? It should be mentioned in the discussion.

To explain the chemical differences between the two trails we added the following paragraph in the discussion section:

Six compounds from the first experiment were not detected in the second: (E)-β-ocimene, trans sabinene hydrate, terpinolene, nonan-1-ol, β-elemene and geranyl α-terpinene. Three compounds were only detected in the second experiment: hexanal, trans-carveol and valencene. With the exception of valencene, none of these compounds exceeds 0.05% and most often is less than 0.01%.  The valencene is an indicator of advanced maturity in sweet orange (Elston et al., 2005) and suggested that the differences could be likely explained by the two-month difference in the sampling date between the two experiments. The cultivar effect could also be a factor of variation as well as the detection threshold mainly for the compounds with very low proportions (below 0.01%) (Dugo et al., 1994).

We added two references to the list in relation with this additional paragraph:

46. Elston, A.; Lin, J.; Rouseff, R. Determination of the Role of Valencene in Orange Oil as a Direct Contributor to Aroma Quality. Flavour Fragr. J. 2005, 20, 381–386; DOI:10.1002/ffj.1578.
47. Dugo, G.; Verzera, A.; d’Alcontres, I.S.; Cotroneo, A.; Trozzi, A.; Mondello, L. On the Genuineness of Citrus Essential Oils. Part XLIII. The Composition of the Volatile Fraction of Italian Sweet Orange Oils (Citrus sinensis (L.) Osbeck). J. Essent. Oil Res. 1994, 6, 101–137; DOI:10.1080/10412905.1994.9698342.

 

2. Some minor expressions should be cautious. E.g, in 2.2.1 Raw materials, "Three lots of five fruits" and in 3.2 below Table 2, in the sentence 'On the other hand,......', '...of the essential oil. (Table 1 and Figure 4') should be changed into " ...of the essential oil (Table 2 and Figure 4)

We modified the expressions as follow:

“Three lots of five fruits" by “Three replicates of five fruits”;

“On the other hand” by “Furthermore”.

We changed Table 1 by Table 2 ......

Reviewer 2 Report

I confirmed the authors corrected this appropriately with improved quality. 

Author Response

We thank the reviewer for the evaluation of our manuscript

Reviewer 3 Report

The manuscript entitled "Influence of the rootstock and the ploidy level of the scion and the rootstock on sweet orange (Citrus sinensis) peel essential oil yield, composition and aromatic properties" sets out to investigates the influence of three different rootstocks and level of ploidy of the scion and the rootstock were studied on the yield, composition, and aromatic profile of sweet orange peel essential oil. The authors responded to all of my previous comments in this version of the manuscript. However, I have some minor concerns about the manuscript that should be addressed before recommending it for publication.

• Any abbreviation must be associated with the full name at the first mention in the manuscript such as NIST in line 173
• In the material and methods section, all the software, chemicals, and equipment sources need to be added or completed (Model or version, add city, state, and country) such as in line135.
• The references list should be formatted according to the MDPI journal style.

Author Response

We thank the reviewer for the evaluation of our manuscript and for his suggestions. The answers to his questions or remarks are the following:

• Any abbreviation must be associated with the full name at the first mention in the manuscript such as NIST in line 173

We have checked the manuscript and we associated the full name with the abbreviations at the first mention

• In the material and methods section, all the software, chemicals, and equipment sources need to be added or completed (Model or version, add city, state, and country) such as in line 135.

We have completed in material section the information of each source (changes were done except for « Target compounds » which is a combination (Model or version, add city, state, and country) except for Excel and Access software.

• The references list should be formatted according to the MDPI journal style.

We have carefully checked all the references in the list and made the necessary changes to bring them into the format of the journal

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

This manuscript entitled … evaluated the effect of rootstock and ploidy level on the composition of orange peel essential oils. [Results and some positive findings]. However, a few but critical weaknesses in the experimental design and data analysis undermined the scientific soundness of this research.

First, in the first experiment three rootstocks (Carrizo citrange, Pomeroy trifoliate and FLHORAG1) were used to evaluate the rootstocks’ effect on essential oil yield, composition and aromatic profile. However, the first two rootstock are diploid, the third one is tetraploid. According to the results, no significant differences existed between the two diploid rootstocks, but there was significant differences between Carrizo and FLHORAG1 on essential oil yield. It is very difficult to explain either the ploidy level or genotypes of rootstock itself caused the differences.

Second, in introduction about grafting ‘Although grafting exist….., a fungi disease [2]’ is not incorrect. Grafting is a very ancient technique used in Citrus. According to the research of Zhaoji Zhou, the article entitled ‘Origin and development of grafting in ancient China’ was published in 1994 on the magazine ‘Studies in the History of Natural Sciences’ 13(3): 264-272. It was reported that tangerine was firstly grafted onto trifoliate orange in 3000 B.C. in an ancient Chinese literature.

Third, sweet orange peel essential oils were obtained by using hydrodistillation. It is well-known that the disadvantages of the hydrodistillation method were long extraction time, possible chemical changes in the structures of terpenes, and the loss of some polar molecules owing to the applied heat. These factors could possibly result in the generation of certain chemicals shown in Table 1. For example, trans-sabinene hydrate is likely a product of sabinene hydration in a heated acid aqueous condition; cis/trans-limonene-1,2-epoxide could be an oxidation product of limonene. In addition, polar molecules commonly reported in orange essential oils such as aldehydes (hexanal, octanal, dodecanal), ketones (carvone, perillaldehyde), alcohols and phenols (octanol, thymol), and esters (citronellyl butyrate) were not detected probably due to a lost in aqueous phase. Generally, the distilled essential oils can not represent the natural composition of original peel oil. The closest sample to a natural oil should be obtained using cold-pressing method.

Fourth, do not quite understand why a GC-FID apparatus was used while the authors already had GC-MS. In the two tables, almost all compounds were identified using RI and MS identification. Were GC-FID used only for calculation of RIs on two different columns? However, the parameters such as flow rate and inner diameter were different between GC-FID and GC-MS, therefore, retention times on the two systems were incomparable, and RIs calculated on GC-FID could not be used on GC-MS. Other issues as described in the method part might explain why some common volatiles not detected. For instance, Line 151-152, oil samples were diluted at a proportion of 11.75/500 which was almost 1/45; then in Line 145, the injected samples were split by 1/60. These operations resulted in a 1/2700 dilution of the oil. No doubt lots of medium-low content were not identified due to very low responses.

   In addition, it would be better to change the Roman number into Arabic number in Line 37 and 42 in Introduction. And the why you studied the essential oil should also be noted in introduction.

Reviewer 2 Report

This is a unique and pioneer work for the citrus industry that sheds light on the hidden correlation of ploidy level and scion/rootstock combination.  
Some experienced farmers and scientists have proposed that rootstock affects the fruit quality of scion, but it has not been determined in detail.

This study is not the first to try to clarify these attractive relationships between scion/rootstock; however, the authors designed several combinations of the different ploidy scion/rootstock to verify the hypothesis, then showed quite exciting relationships. The used materials and experimental design are explicit, and the data interpretation is rational. 

Though we have no concrete idea how to interpret the results so far, the demonstrated results would call the new research theme. 
Thus, I strongly encourage this.

I also encourage the authors to check the following and correct them appropriately for further improvements.

L100: deliciosa or reticulata?
L102-107: Briefly describe the origin of those 4X plants, or cite corresponding references.

L270: I wonder Table 1 might be Table 2.

Table 1: Describe for IK A and IK P in the legend.

Figure 2: Explain the PCA position and arrow for chemicals. Also, please explain the meaning/effect/correlation of chemicals in the PCA plot in the results section.

Confirm the journal name of reference 2, 10, 11,  
Reference 13: Capitalize to 'MALTAISE DEMI SANGUINE'
Please double-check for the reference format.

Reviewer 3 Report

The manuscript entitled "Influence of the rootstock and the ploidy level of the scion and the rootstock on sweet orange (Citrus sinensis) peel essential oil yield, composition and aromatic properties" sets out to investigate the influence of three different rootstocks and the level of ploidy of the scion and the rootstock were studied on the yield, composition, and aromatic profile of sweet orange peel essential oil. The authors concluded that the rootstock genotype influenced the peel essential oil yield of the fruit, whereas the level of ploidy of the rootstock seemed to have no influence or the level of ploidy of the scion. The study is on a topic of relevance and general interest to the readers of the journal. I found the paper to be overall well written. However, I have several concerns about the manuscript that should be addressed.

• The authors are highly recommended to avoid using a personal pronoun (e.g., We, our, etc.); they can use the third party in the past tense's passive voice.
• The authors need carefully to read the manuscript to correct typos and grammars to improve the manuscript.
• In the abstract change XIX to 19 (line 37) and XX to 20 (line 42) because not all the readers are familiar with the Roman numbers.
• In the material and methods section, all the sources of software, chemicals, and equipment need to be added or completed (Model or version, add city, state, and country).
• Tables 1 and 2, mean, sd, and groups must be in one column (Mean±sd followed by the group letter), any abbreviation in the table should be defined in the table footnote, not in the table title, the authors need to define in the table footnote what * refer to.
• Figures 2 and 4, Change Dim1 and Dim2 to PCA1 and PCA2
• The references list should be formatted according to the MDPI journal style.