Evaluation of Image-Based Phenotyping Methods for Measuring Water Yam (Dioscorea alata L.) Growth and Nitrogen Nutritional Status under Greenhouse and Field Conditions

Round 1

Reviewer 1 Report

This manuscript showed yam scientists a reality that sets image-based phenotyping techniques on the evaluation of traits relevant for tuber yield or growth of aboveground of yam. Originality of this manuscript is these results generated from glasshouse with well regulated environment condition and field trials, especially in real yam cropping land where people cultivate yam as a staple food. I really hope that this methods will be more polished, simplified, and disseminated into research institutes to study on yam in NARSs in West Africa.  

In P16 Line 632 to 635, authors discussed about the reason of low tuber yield of C18 of water yam. If the growth of aboveground was still in vigorous or not in senescence stage around November, please guess of the possibility of earliness of the variety C18. C18 may be late mature variety and it may not adopt in the trial area. This is my comment.

Author Response

This manuscript showed yam scientists a reality that sets image-based phenotyping techniques on the evaluation of traits relevant for tuber yield or growth of aboveground of yam. Originality of this manuscript is these results generated from glasshouse with well regulated environment condition and field trials, especially in real yam cropping land where people cultivate yam as a staple food. I really hope that this methods will be more polished, simplified, and disseminated into research institutes to study on yam in NARSs in West Africa.  

Thank you for your encouraging words, which we take as a motivation to do so.

In P16 Line 632 to 635, authors discussed about the reason of low tuber yield of C18 of water yam. If the growth of aboveground was still in vigorous or not in senescence stage around November, please guess of the possibility of earliness of the variety C18. C18 may be late mature variety and it may not adopt in the trial area. This is my comment.

We removed this discussion as this was considered to be not relevant for this manuscript (see answer to reviewer 2). But we have added l 207 that tubers were harvested when the plants were senescent. C18 is indeed a late cultivar.

Reviewer 2 Report

The ms describes an approving the image-based methods for measuring growth parameters and nitrogen content in water yam. Authors found a good effectiveness of digital image analysis the leaf projection area to assess shoot biomass and total leaf surface in greenhouse experiment which appeared in greater degree on early stages of plant growth. The ‘triangular greenness index’ was found non-informative for measurement of leaf nitrogen. Before this work is published authors should consider the following comments:   

Authors masterly use the modern methods of image analysis and plant sampling and obtained interesting data but data presentation and discussion are problematic. Almost all sections of ms including introduction, results, discussion and conclusions contain many aspects of plant biology which do not concern the main idea of the ms. The goal of the paper is the evaluation of method for measuring the plant traits but not the study of impact of nitrogen on growth, influence of emergence on biomass or tuber yield and so on (Discussion L.584). As a result, the text is full of extra unnecessary information. It is not important for goal of the paper that assessing traits are relevant “for tuber yield formation”. This information should be strongly reduced in Introduction as well as a description of yams taxonomy and planting. However, the information about the use of image-phenotyping methods in measuring of plant biomass must be improved and strongly expanded.   

Describing of Methods is definitely too large and too comprehensive and should be reduced. At the same time, it is unclear how authors did measure real value of the total leaf surface (cm2 per plant). There is no explanation for this in Methods. Besides authors should explain “triangular greenness index (TGI)” and “SPAD” methods more clearly.

According to the objective of ms authors should describe the effectiveness of image-phenotyping method but neither the features of the plant growth nor the regularities of nitrogen accumulation (Results L.344-419, Discussion L.584-585, L.591-596, L.618-635). In this case, the reader is not interested, why the growth of this plant was scarce. One is interested in correctness of the image analysis for the measurements of plant growth independently of influencing factors. The entire section 4.2.1 as well as section 4.2.2. is dedicated to the reasons of pure growth of yam instead the discussion of possibility of use image-based method for measuring yam growth in the field.

In conclusions authors have to stress more clearly that image-based analysis is suitable for early stages of plant growth but TGI was not shown to be informative for leaf N content.   

Some minor comments:

Figure 6 duplicates the table 2 and one of them should be deleted.

Figure 7 is not correctly visible in ms

Table 3 is not correctly visible in ms

L. 376 “days weeks”

L. 426-427 should be moved from Results to Discussion

L. 441-444, L. 484-485 should be moved from Results to Discussion

L.508-511 should be moved from Results to Methods

L.591-596 should be moved from Discussion to Methods

Author Response

Comments and Suggestions for Authors

The ms describes an approving the image-based methods for measuring growth parameters and nitrogen content in water yam. Authors found a good effectiveness of digital image analysis the leaf projection area to assess shoot biomass and total leaf surface in greenhouse experiment which appeared in greater degree on early stages of plant growth. The ‘triangular greenness index’ was found non-informative for measurement of leaf nitrogen. Before this work is published authors should consider the following comments:   

Thank you.

Authors masterly use the modern methods of image analysis and plant sampling and obtained interesting data but data presentation and discussion are problematic. Almost all sections of ms including introduction, results, discussion and conclusions contain many aspects of plant biology which do not concern the main idea of the ms. The goal of the paper is the evaluation of method for measuring the plant traits but not the study of impact of nitrogen on growth, influence of emergence on biomass or tuber yield and so on (Discussion L.584). As a result, the text is full of extra unnecessary information. It is not important for goal of the paper that assessing traits are relevant “for tuber yield formation”. This information should be strongly reduced in Introduction as well as a description of yams taxonomy and planting. However, the information about the use of image-phenotyping methods in measuring of plant biomass must be improved and strongly expanded.   

Thank you for this comment. We deleted the parts that you considered out of the topic from the introduction, results and discussion.

We have improved and expanded information on image-phenotyping methods (TGI) as requested (see below).

However, we think that it is important to work on traits relevant for tuber yield formation, as this is at the end what needs to be improved.

 

Describing of Methods is definitely too large and too comprehensive and should be reduced. At the same time, it is unclear how authors did measure real value of the total leaf surface (cm2 per plant). There is no explanation for this in Methods. Besides authors should explain “triangular greenness index (TGI)” and “SPAD” methods more clearly.

We reduced as much as possible the description of methods by leaving out extra information, and rewording sentences. However, the description of our materials and methods remains comprehensive so that other authors can repeat our experiments.

We describe now the total leaf surface as follows (ll 140-143)

“All leaves of a given plant were spread on a blue background, then nadir images were taken with the indoor imaging station described in 2.3.1 and the images were processed using the methodology described in section 2.3.3”

We describe now SPAD as follows (ll 151-157)

“A SPAD meter (SPAD-502, Minolta Corporation, 130 Ramsey, NJ, USA) was used to estimate the chlorophyll content which is often positively related to the leaf N content [26,27,28]. The SPAD meter measures light absorption by the leave at 650 nm, which is the wavelength at which chlorophylls a and b absorb light and at 950 nm, which is a wavelength that is theoretically not absorbed by the chloroplast, and it calculates a SPAD value [27]. High amount of chlorophyll in a leaf leads to high absorption in the red spectral range and therefore to an intensive, green coloration of the leaf.”

And we describe TGI as follows (ll 291-302)

“The triangular greenness index (TGI) makes use of the differences between reflectance in the red (r), green (g) and blue (b) camera channels by calculating the area spanning between the three points in the spectra as follows [10]:

TGI = -0.5x[(W670-W480)x(Rr-Rg)-(W670-W550)x(Rr-Rb)]                      Equation [1]

With W670, W480, W550 being the center wavelengths in nm; Rr, Rg, and Rb are the reflectance values for the three camera bands (r, 630–690 nm, g, 520–600 nm and b, 450–520 nm), respectively. In the glasshouse the TGI values were derived from nadir-recorded images of separated leaves and averaged at plant level (Figure 2e). Due to the calculation scheme of this index, dark green leaf regions have rather low TGI values, whereas yellow-green leaf regions are characterized by high TGI values (i.e., high chlorophyll content leads to low TGI value).”

According to the objective of ms authors should describe the effectiveness of image-phenotyping method but neither the features of the plant growth nor the regularities of nitrogen accumulation (Results L.344-419, Discussion L.584-585, L.591-596, L.618-635). In this case, the reader is not interested, why the growth of this plant was scarce. One is interested in correctness of the image analysis for the measurements of plant growth independently of influencing factors. The entire section 4.2.1 as well as section 4.2.2. is dedicated to the reasons of pure growth of yam instead the discussion of possibility of use image-based method for measuring yam growth in the field.

Most of the above-mentioned parts of the initial manuscript have been deleted from the text. The discussion has been largely rewritten. The results pertaining to the deleted parts are shown in the supplementary materials, so that the interested reader can consult them.

In conclusions authors have to stress more clearly that image-based analysis is suitable for early stages of plant growth but TGI was not shown to be informative for leaf N content.   

We hope that the manuscript is now clearer. We now mention in the perspective that the presented approach should be validated for longer growth periods in the field. We realize that the relationship between TGI and N content is a complex one, but we suggest that more research should be done to see whether TGI could be used as a tool for improving N management in yam in the future.  

Some minor comments:

Figure 6 duplicates the table 2 and one of them should be deleted.

This statement is only true for the first line in what has become table 1. This table shows also how the view angle and the number of images relate to the measured traits. Although multiple perspectives are standard for indoor imaging, they are practically not feasible under field conditions. Given the growth habit of yam it was not evident that the sole nadir view would already show excellent results and that additional views would not add much information. This is for us an important result. Figure 4 (former figure 6) shows the distribution of points as well as the correlation model for the nadir view. We consider both illustrations as complementary and this is why we would prefer to keep both table 1 and figure 4.

Figure 7 is not correctly visible in ms

Sorry about that, we have enlarged it (this is now figure 5).

Table 3 is not correctly visible in ms

Sorry about that, we have transferred this table to the supplementary materials.

1. 376 “days weeks”

Thank you, weeks was deleted, and this text was moved to the supplementary materials.

1. 426-427 should be moved from Results to Discussion

This was done.

1. 441-444, L. 484-485 should be moved from Results to Discussion

This was done.

L.508-511 should be moved from Results to Methods

We would like to keep this text on results in the results section.

L.591-596 should be moved from Discussion to Methods

This text was deleted.

Round 2

Reviewer 2 Report

Authors have added required changes to the text and figures according to reviewer’s comments. The ms has been essentially improved in its structure and style as well as in English language.