The Use of Near-Infrared Imaging (NIR) as a Fast Non-Destructive Screening Tool to Identify Drought-Tolerant Wheat Genotypes
, Domenico De Paola 1, Angelo Petrozza 2, Stephan Summerer 2, Francesco Cellini 2, Domenico Pignone 1,* and Michela Janni 3,*
Round 1
Reviewer 1 Report
The manuscript brings good results that could support future research.
Lines 111-117: there is no information on how the analysis is done. You should inform important details on how to obtain the data used in this analysis.
Other criticizes I could make is on the figures and supplementary material.
On the supplementary material, it seems that the term sd is duplicated. Please correct it. On the figures, I think you should better label de information on the x and y-axis. Everything else looks fine.
Author Response
Lines 111-117: there is no information on how the analysis is done. You should inform important details on how to obtain the data used in this analysis.
Answer Thanks for suggesting this improvement, we have added the following paragraph.
Line 131-136
Answer For this work segmentation of the NIR images consisted first of adaptive thresholding (box size 10 x 10 pixels, threshold difference of 10). Artifacts of less than 3 pixels were eliminated with the fill function by identifying a region of interest (ROI) corresponding to the area occupied by plants that was used to exclude artifacts from the imaging chamber. The intensity values of the remaining plant pixels were used for histogram analysis
Other criticizes I could make is on the figures and supplementary material.
On the supplementary material, it seems that the term sd is duplicated. Please correct it.
Answer Done
On the figures, I think you should better label de information on the x and y-axis. Everything else looks fine.
Answer- We have modified the figure as suggested, however since was impossible to make readable the legend of the x axis, we have removed the label of the x axis from Fig. 2 and Fig. 3. We have included in the text Table 2 that was formerly included in the supplementary material,
Reviewer 2 Report
The manuscript presents a method to identify drought tolerant genotypes for breeding program by using near-infrared imaging technology. The idea is interesting and has application potentials.
However, the scientific contribution of this manuscript is not strong, because similar ideas and approaches have been reported in literature. The first suggestion to the authors is to describe or to enhance in more details the novelty of their approach by comparing their proposed methodology with those previous methods in literatures.
Technically, some processing in the method is arguable. For example, the authors claim “Water absorption bands are typical at 1450, 1930 and 127 2500nm of the NIR spectrum, …… allows measurement at the 1450nm water absorption band.” It seems that the authors consider the water absorption band as the indicator for wheat leaf water level. But the water absorption is actually caused by atmospheric transmission loss, and no evidence has bee given so far that the water absorption bands are associated with surface water level. This put a serious doubt on the technique correctness of the manuscript.
The presentation of the manuscript could also be improved. For example, the axis labels of Figure 2 are so obscure and no texts can be identified. The sentence “… we defined “WET pixels” those showing a luminance range 50 ≤ L ≥ 80 and representing well-watered plant parts; conversely, all pixels with a luminance value L > 80 were considered DRY pixels” is not understandable, and it seems that “50 ≤ L ≥ 80” should be “50 ≤ L ≤< 80”, and so on.
Author Response
The first suggestion to the authors is to describe or to enhance in more details the novelty of their approach by comparing their proposed methodology with those previous methods in literatures.
Answer. We thank the reviewer for the positive comment. We agree we have not underlined enough the novelty of the work. A significant literature is reported on the use of NIR imaging to perform plant phenotyping to recover the hydration of plant leaves. The actual novelty of this work is the application of NIR technology to High Throughput Phenotyping for scoring segments of plant genetic resources collections. NIR technology has been used with hand-held instruments to analyze leaf water content, in a very time consuming procedure based on few sample points in the plant. Our approach records water content of the entire plant in around one minute, a time several folds faster the previous methods. This manuscript paves the way for a novel approach for genotype selection, unlocking the great poteintial of genetic resources in plant breeding. We have included the following statements in the manuscript together with an extended conclusion of the presented work.
Line 231-243
The results of this experimental approach have clearly demonstrated that application of NIR technology for High Throughput Phenotyping can be successfully employed in selecting useful genotypes from germplasm collections. NIR technology has been routinely used with hand-held instruments to analyze leaf water content, in a very time consuming procedure based on few sample points in the plant [28]. Here the use of a NIR camera allows the rapid evaluation of plant water status in a fast and non destructive way, allowing to monitoring one and the same plant over the time domain. This evenience reduces the bias introduced by individual variation while exhamining the response to an external stimulus.
Moreover, if HTP data could be associated with genomic data for single genotype, this approach will prompt the identification of genetic traits potentially associated to the desired character more rapidly and precisely in comparison with costly and time-consuming field experiments based on association approach, such as GWAS.
Technically, some processing in the method is arguable. For example, the authors claim “Water absorption bands are typical at 1450, 1930 and 127 2500nm of the NIR spectrum, …… allows measurement at the 1450nm water absorption band.” It seems that the authors consider the water absorption band as the indicator for wheat leaf water level. But the water absorption is actually caused by atmospheric transmission loss, and no evidence has been given so far that the water absorption bands are associated with surface water level. This put a serious doubt on the technique correctness of the manuscript.
Answer: Actually liquid water has absorption peacks at 1450 nm and 1930 nm, The absorption can be observed in the atmosphere, in transmitted light configuration, or in reflected ligh configuration, as in the case of the light reflected by a leaf. This is the principle on which many hand-held or field instruments work. We rephrased as follow:
Line 131-line 136
Typical water absorption bands in the Near Infrared spectrum are at 1450nm and 1930nm, thus the Scanalyzer NIR camera, being sensitive in the 900 – 1700nm range allows measurement of the drop in foliar reflectivity due to the 1450nm water absorption band. An increased level of absorètion will result in a reduced level of reflection in the NIR spectrum, thus allowing to estimate leaf water content
The presentation of the manuscript could also be improved.
For example, the axis labels of Figure 2 are so obscure and no texts can be identified.
The sentence “… we defined “WET pixels” those showing a luminance range 50 ≤ L ≥ 80 and representing well-watered plant parts; conversely, all pixels with a luminance value L > 80 were considered DRY pixels” is not understandable, and it seems that “50 ≤ L ≥ 80” should be “50 ≤ L ≤< 80”, and so on.
Answer-Thanks for the suggestion.
We have rephrased as follow:
Line 176-179
We defined “WET pixels” those showing a luminance (L) range of 50 ≤ L ≤ 80 and representing plant parts richer in water; conversely, we named DRY pixels all pixels with a luminance value L > 80, thus representing plant parts with a lower water content.
Reviewer 3 Report
I have reviewed the manuscript entitled “The use of near-infrared imaging (NIR) as a fast non-destructive screening tool to identify drought-tolerant wheat geno- 3 types” and found it novel. The statistics used for the experiment is well suitable and the data is presented in excellent manner. However, there are several typing and grammatical mistakes that need to be corrected. Furthermore, there are some other technical suggestions and questions if addressed will further improve the quality of the manuscript.
- I suggest to link the sentence “To ensure food security while improving agriculture sustainability is the mission of the upcoming decades” to drought .
- In abstract section, your results are emphasizing on methods rather than findings. I suggest, to presents your major findings.
- Arrange the keywords in alphabetic order.
- Explain the abbreviation like CNR and NIR etc. when used for the 1st
- How did you select 137 genotypes out of 411? What were the selection criteria?
- Why did you add 1.2kg soil to 2L pots knowing the fact that bulk density of the soil is greater than 1.
- How did you maintain 100% Field Capacity till tillering stage?
- I cannot see any discussion of your results in the manuscript why?
- The conclusion section needs proper heading and revision.
- Format the references as per journal requirement.
Author Response
I have reviewed the manuscript entitled “The use of near-infrared imaging (NIR) as a fast non-destructive screening tool to identify drought-tolerant wheat geno- 3 types” and found it novel. The statistics used for the experiment is well suitable and the data is presented in excellent manner. However, there are several typing and grammatical mistakes that need to be corrected.
Answer- Thanks for the positive comment. We apologies for the typing and mistakes, we have gone thorough the entire document and we hope now is suitable for the publication.Furthermore, there are some other technical suggestions and questions if addressed will further improve the quality of the manuscript.
Answer- Thanks to Reviewer 3 for the comments that will surely improved the quality of the manuscript
I suggest to link the sentence “To ensure food security while improving agriculture sustainability is the mission of the upcoming decades” to drought .
We have rephrased as follow:
Line 17-18: Due to climate change, many agricultural areas will face shortage in water availability; thus the mission of the upcoming decades is to ensure food security while improving agriculture sustainability-
In abstract section, your results are emphasizing on methods rather than findings. I suggest, to presents your major findings.
Answer-We have rephrased according to your suggestion.
Line 23-29: To select contrasting phenotypes within a core set of 149 durum wheat genotypes, belonging to the CNR germplasm collection, under drought stress conditions we studied plant water status gained by NIR imaging. By the application of the present method it was possible to rapidly identify and select a set of putative genotypes higly tolerant to drought, as well as a set of contrasting genotypes, for forther studies and/or for breeding programs. This promising approach is scalable to a larger number of genotypes
Arrange the keywords in alphabetic order.
Answer- Done. durum wheat, genetic resources, NIR, phenotype selection, plant phenotyping
Explain the abbreviation like CNR and NIR etc. when used for the 1st
Done. Line 24
How did you select 137 genotypes out of 411? What were the selection criteria?
Answer-They were randomly selected to try maintain the level of diversity present in the SSD collection, We had to reduce the number of genotypes scored due to the capacity of the platform which cannot host more than 500 pots. We have included this point in the Material and methods section.
Line 91-93
The 137 genotypes were randomly selected maintaining the high level of diversity present in the SSD collection and according to the capacity of the phenotyping platform which cannot host more than 500 pots.
Why did you add 1.2kg soil to 2L pots knowing the fact that bulk density of the soil is greater than
Answer- The density of the mixture we used is much lower than 1, being around 0.65. Moreover the pots are not filled to the rim to let the implant of a disk of the same color and material of the pot to minimize the bias in plant imaging
How did you maintain 100% Field Capacity till tillering stage?
Answer- The LemnaTech Platform automatically weights each pot after measurement and fill the pot with the correct amount of water necessary to re-establish the weigth assigned to that pot. The rationale is that the wheight of the plant is marginal respect to that of the rest of the pot, so the weight loss is due to water loss. We have included the following sentence:
Line 104
Pots were automatically watered daily to saturation to avoid soil moisture deficit.
I cannot see any discussion of your results in the manuscript why?
Answer- The discussion has been developed along with the exposition of the results
The conclusion section needs proper heading and revision.
Answer- We have rephrased the conclusions as follow:
Line 231-243
The results of this experimental approach have clearly demonstrated that application of NIR technology for High Throughput Phenotyping can be successfully employed in selecting useful genotypes from germplasm collections. NIR technology has been routinely used with hand-held instruments to analyze leaf water content, in a very time consuming procedure based on few sample points in the plant [28]. Here the use of a NIR camera allows the rapid evaluation of plant water status in a fast and non destructive way, allowing to monitoring the one and the same plant over the time domain. This evenience reduces the bias introduced by individual variation while exhamining the response to an external stimulus.
Moreover, if HTP data could be associated with genomic data for single genotype, this approach will prompt the identification of genetic traits potentially associated to the desired character more rapidly and precisely in comparison with costly and time-consuming field experiments based on association approach, such as GWAS.
Format the references as per journal requirement.
Answer- done
Round 2
Reviewer 2 Report
The authors have addressed my concerns by carefully rewriting some parts of the manuscript. The following suggestions are provieded to further improve the manuscript.
- A proper legend can be added to the right side of Figure 2.
-
Abbreviations can be checked to fit domain conventions and be explained properly, e.g., Nir, se, SSD etc.
- Table 2 is too long, and can be re-designed into a proper presentation.
Author Response
Thank you for suggestions
A proper legend can be added to the right side of Figure 2.
The labels of Fig.2 have been modified together with figure's legend
Abbreviations can be checked to fit domain conventions and be explained properly, e.g., Nir, se, SSD etc.
Done
Table 2 is too long, and can be re-designed into a proper presentation.
Table 2 has been reshaped in a shorter form
Moreover, two email addresses have been corrected, the legend of Figure 3 has been modified, and a few mispelled words corrected
