Sand Priming Promotes Seed Germination, Respiratory Metabolism and Antioxidant Capacity of Pinus massoniana Lamb.
Round 1
Reviewer 1 Report
Only praise for the work, especially Figure 4. I am of the opinion that sand priming research and his influence of seed biology should be continued in this species in nursery conditions and in the field (afforestation by sowing). You have not written a conclusion and it must be added, otherwise I cannot accept the paper for publication.
Author Response
Q1: Only praise for the work, especially Figure 4.
Response: Thanks for your approbation for our work and we will revise our manuscript carefully and looking forward to achieving your approval.
Q2: I am of the opinion that sand priming research and his influence of seed biology should be continued in this species in nursery conditions and in the field (afforestation by sowing).
Response: Thanks a lot for your good suggestion. The field experiment was synchronously conducted with laboratory germination. Considering that there was too much germinated data, the field part was not integrated into the original manuscript. In the revised manuscript, the corresponding contents had been added to the parts of abstract (Lines 18-20, Page 1), methods (Lines 132-139, Page 3), results (Lines 198-209, Page 5) and Table 2 (Page 5).
Q3: You have not written a conclusion and it must be added, otherwise I cannot accept the paper for publication.
Response: Thanks for your reminding. According to your advice, we have supplemented the following contents “In conclusion, our results show that sand priming could promote seed germination and field emergence, antioxidant ability and seed respiration of Pinus massoniana. Significant differences in germination rate, mean germination time, the activity of antioxidases (POD, SOD, CAT), endogenous phytohormone level (GA, IAA, ABA), and seed respiration were observed after sand priming treatment. Sand as solid matrix to prime the seeds of Pinus massoniana, may contribute to break seed dormancy and improving seed vitality of Pinus massoniana, on a deeper layer, which will also provide an effective pretreatment method for the afforestation project.” to Conclusion part at Lines 357-365, Pages 12.
Reviewer 2 Report
Line 68 I recommend changing 'elucidated' to 'evaluated' it will change reception
In the methods section, suggests expanding the description of the methods used. "...the mean germination time was calculated by the method of [11]" this will make it easier to read and will not require additional searching by the reader. This paper has not been published in the open access.
Lina 118-120 as mentioned above.
Data analysis - explain what is the first and second factor.
Table 3 - under the table please add an explanation of the marks: IMT, OMR, COP and RGT
Change the diagrams in the text to more readable, larger ones. Descriptions are currently not visible.
Author Response
Q1: Line 68 I recommend changing 'elucidated' to 'evaluated' it will change reception
Response: Thanks for your advice. We have changed the 'elucidated' to 'evaluated'. Meanwhile we revised some contents to make the paper more logical. Therefore, the sentence “In this study, the effects of sand priming on seed germination, respiratory metabolism and antioxidant capacity of Pinus massoniana were elucidated. To further clarity how it affected,” was changed into “Previous researches had proved the sand priming was a valid pretreatment to promote seed germination in some field crops. Therefore, a hypothesis was proposed that whether sand priming could be applied to promote seed germination of Pinus massoniana. In order to evaluate the effects of sand priming Pinus massoniana,” at Lines 72-76, Page 2.
Q2: In the methods section, suggests expanding the description of the methods used. "...the mean germination time was calculated by the method of [11]" this will make it easier to read and will not require additional searching by the reader. This paper has not been published in the open access.
Response: Thanks for your reminding. Based on your suggestions, “The germination rate, germination index and vigor index were determined according to our previous paper [20], and the mean germination time was calculated by the method of [11]” had been changed into “Germination rate (GR), germination index (GI), vigor index (VI)[20] and the mean germination time (MGT) [11]were calculated according to the following formulas:
GR= The number of germinated seeds within the 12 days / The total number of seeds × 100% GI=∑ (Gt/ Dt), here Gt is corresponding number of seeds germinated in the t day; Dt is time corresponding to Gt in days.
VI = GI ×S, here GI refers to germination index. S is the average length of 10 seedlings.
MGT = ∑TiNi/∑Ni, here Ni referred to the number of the new germination seeds in day Ti)” at Lines 116-127, Page 3.
Q3: Lina 118-120 as mentioned above.
Response: Thanks for your reminding. Based on your suggestions, “The activities of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) of sand-primed seeds and non-primed seeds were assessed according to the research [22]. Succinate dehydrogenase (SDH) activity was determined by the method in [23]. The content of Malondialdehyde (MDA) and proline was assayed by the reference [24].” had changed into “Superoxide dismutase (SOD) activity was measured by determining the inhibition to photochemical reduction of Nitroblue tetrazolium as monitored at 560 nm; catalase (CAT) activity was measured by the absorbance decrease at 240 nm based on the H2O2 decomposition at 25°C; peroxidase (POD) activity was measured by monitoring the increase in absorbance at 470 nm due to guaiacol oxidation at 25°C [22]. Succinate dehydrogenase (SDH) activity was determined by mg of SDH per g protein an hour using the extinction coefficient 1.03x103/M/cm at 420 nm [23]. The content of Malondialdehyde (MDA) was calculated by the formula “μmol MDA g−1 FW = 6.45 (OD532 − OD600) − 0.56 * OD450 ”; proline content was assayed by the spectrophotometer at 520 nm [24].” at Lines 153-165, Page 4.
Q4: Data analysis - explain what is the first and second factor.
Response: The effect of treatment and variety is recognized as the first and second factor. The sentence was supplemented at Lines 194-195, Page 5.
Q5: Table 3 - under the table please add an explanation of the marks: IMT, OMR, COP and RGT
Response: Thanks for your reminding. We have supplemented the explanation of IMT, OMR, COP and RGT at Lines 232-234, Page 7 as follows:
“IMT: increased metabolism time; OMR: oxygen metabolism rate; COP: critical oxygen pressure; RGT: relative germination time.”
Q6: Change the diagrams in the text to more readable, larger ones. Descriptions are currently not visible.
Response: Thanks for your reminding. Based on your recommendation, Figure 1, Figure 2 and Figure 3 had been updated in a new version to make the information more visible.
Reviewer 3 Report
The manuscript contains too much shortcomings in methodology and so many flaws that in the current form should not be under any consideration. Introduction part does not explain properly why the study was performed. It lacks hypothesis and description of the aim of the study. There is no information why seeds of 3 varietis were used. I am not sure what was the real material - seeds or seedlings, such basic information are absent. There is no information on pine seeds dormancy and the impact of storage conditions on seed dormancy status. Authors should discuss and compare their data to data obtained rather for seeds of trees, not cereals. Some detailed comments particularly on M&M are included in the manuscript pdf version. M&M section is of quality unacceptable for any journal - methods need to be desribed in details, not only by references.
Comments for author File: 
Comments.pdf
Author Response
Q1: The manuscript contains too much shortcomings in methodology and so many flaws that in the current form should not be under any consideration.
Response: Thank you for pointing out our problems of our manuscript. We will revise our manuscript carefully and looking forward to achieving your approval.
Q2: Introduction part does not explain properly why the study was performed. It lacks hypothesis and description of the aim of the study.
Response: Thanks for your reminding. According to your comments, we had supplemented the sentence “Previous studies had proved the sand priming was a valid pretreatment to promote seed germination in some field crops. Therefore, a hypothesis was proposed that whether sand priming could also be applied to promote seed germination of Pinus massoniana” in the section of Introduction at Lines 72-76, Page 2.
Q3: There is no information why seeds of 3 varietis were used. I am not sure what was the real material - seeds or seedlings, such basic information are absent.
Response: Thanks for your questions. Laoshan forestry centre preserves the most important germplasm resources of Pinus massoniana in China. The three varieties in seed orchard of masson pine are gradually improved and they have been widely used in genetic improvement and afforestation. Therefore, they are the typical representatives of masson pine varieties. The sentence “Laoshan forestry centre preserves the most important germplasm resources of Pinus massoniana in China. The above three varieties in seed orchard of masson pine are gradually improved and they have been widely used in genetic improvement and afforestation” was added at the section of Plant materials. The seeds of masson pine varieties were used as experimental materials. This information had been shown in the section of Plant materials such as the sentences “Seeds were stored at 4℃ for 3 months before experiment” (Lines 90-93, Page 2).
Q4: There is no information on pine seeds dormancy and the impact of storage conditions on seed dormancy status. Authors should discuss and compare their data to data obtained rather for seeds of trees, not cereals.
Response: Thank you for your reminding. To break dormancy, seeds were put into zippered plastic bags and subjected to moist chilling at 4℃ at 35% moisture content for 60 d in the dark. The initial germination rate ranged from about 10-40% while was increased to approximately 20-65% after moist chilling. This information was added at the part of materials (Lines 95-98, Pages 2-3). This is the standard treatment used in operation practice. So, after moist chilling, seeds were stored at 4℃ for 3 months before experiment at Line 98, Page 3.
Q5: Some detailed comments particularly on M&M are included in the manuscript pdf version. M&M section is of quality unacceptable for any journal - methods need to be desribed in details, not only by references.
Response: Thanks for your reminding. Based on your comments on M&M section, we have carefully refined this part as follows:
- “The germination rate, germination index and vigor index were determined according to our previous paper [20], and the mean germination time was calculated by the method of [11]” had been changed into “Germination rate (GR), germination index (GI), vigor index (VI)[20] and the mean germination time (MGT) [11]were calculated according to the following formulas:
GR= The number of germinated seeds within the 12 days / The total number of seeds × 100% GI=∑ (Gt/ Dt), here Gt is corresponding number of seeds germinated in the t day; Dt is time corresponding to Gt in days.
VI = GI ×S, here GI refers to germination index. S is the average length of 10 seedlings.
MGT = ∑TiNi/∑Ni, here Ni referred to the number of the new germination seeds in day Ti)” at Lines 116-127, Page 3.
- “The activities of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) of sand-primed seeds and non-primed seeds were assessed according to the research [22]. Succinate dehydrogenase (SDH) activity was determined by the method in [23]. The content of Malondialdehyde (MDA) and proline was assayed by the reference [24].” had changed into “Superoxide dismutase (SOD) activity was measured by determining the inhibition to photochemical reduction of Nitroblue tetrazolium as monitored at 560 nm; catalase (CAT) activity was measured by the absorbance decrease at 240 nm based on the H2O2 decomposition at 25°C; peroxidase (POD) activity was measured by monitoring the increase in absorbance at 470 nm due to guaiacol oxidation at 25°C [22]. Succinate dehydrogenase (SDH) activity was determined by mg of SDH per g protein an hour using the extinction coefficient 1.03x103/M/cm at 420 nm [23]. The content of Malondialdehyde (MDA) was calculated by the formula “μmol MDA g−1 FW = 6.45 (OD532 − OD600) − 0.56 * OD450 ”; proline content was assayed by the spectrophotometer at 520 nm [24].” at Lines 153-165, Page 4.
3.” Endogenous gibberellin A1 (GA1), abscisic acid (ABA) and indole-3-acetic acid (IAA) were measured by LC-ESI-MS/MS [25].” had changed into “The cryopreserved seed embryos of Pinus massoniana were ground to powder. Based on the 1:10 ratio (M/V), 0.5 g of powder was added into 5 ml of 80% methanol extract in the centrifuge tube. During this period, vortex once every half an hour and repeat it 5 times. After stand for 12 h at 4°C in the dark, centrifugation at 12,000 g at 4°C for 20 min was conducted to separate of precipitate. 500 μL of 30% methanol solution was used to dissolve the precipitate. Centrifuge at 12,000 g for 15 min at 4°C to collect the precipitate and store it in the sample vial. Endogenous gibberellin A1 (GA1), abscisic acid (ABA) and indole-3-acetic acid (IAA) were measured by LC-ESI-MS/MS [25]. MS system: ion spray voltage -4,500 V, temperature 550°C, curtain gas 40 psi, Gas 1:50/Gas 2:50. Chromatographic system: HSS T3 liquid chromatography column (100 * 2.1 mm, 1.8 µm), mobile phase A (0.1% formic acid-aqueous solution), mobile phase B (0.1% formic acid-acetonitrile), sample volume = 5 μL.” at Lines 167-179, Page 4.
