Physio-Morphological and Biochemical Trait-Based Evaluation of Ethiopian and Chinese Wheat Germplasm for Drought Tolerance at the Seedling Stage

Round 1

Reviewer 1 Report

The topic of the manuscript is powerful and actual. The study is quite conventional, providing negligible progress in methodology or general knowledge. On the other hand, using a high number of genetic resources of different origin, it may provide some interesting information on variability in responses. In this respect, however, the weak spot of the present version is that the genotypes are not presented with their names, origins, and species, and the authors provide only anonymous names in the text (providing only the list as supplementary files). Moreover, no analyses related to the origin, species, or other characteristics are shown, which limits the value of the presentation. It would be much more convenient if each name contains code with the origin, species – e.g., in Fig. 8, it can provide interesting information. Thus, the authors insufficiently gain the potential of the results obtained.

Nevertheless, the study is well written and provides quite a meaningful discussion. The results are valuable, but a significant revision is needed, as shown by my comments below. I suggest giving the authors a chance to revise the manuscript, appropriately addressing my comments below.

1. 165: It is claimed that in the experiments, “LED fluorescent lamps” were used, which is confusing. Were the lamps fluorescent or LED? If fluorescent, the type, and producer of the fluorescent tubes should be added. In the case of LED, the producer is not essential, but a spectrum, or, at least, the contribution of red and blue (or other) LEDs should be specified.
2. 186-187: This part is confusing: “… as per the instruction of the manufacturer (Yamei Horticulture Co., Ltd Shijiazhuang, Hebei 186 province, China)”. Manufacturer of what? I am in doubt that any manufacturer provides instructions relevant to that kind of study.
3. In the case of total water consumption, I miss information on the leaf area, which may significantly influence the transpiration.
4. It is not clear why the SPAD was measured, as much more precise chlorophyll content analyses were done. It is duplicity unless the authors provide clear arguments justifying the difference in information obtained.
5. In the case of proline content and photosynthetic pigments, it seems that the content was calculated per fresh mass unit. However, it is not clear whether the recalculation per fully turgescent state was done. Simply, the sample that loses more water has a higher concentration of pigments or proline than the sample losing less. So, without recalculation, it is comparing non-comparable values. It must be re-calculated or deleted – it cannot be used in the paper, as it is an apparent artifact!!!
6. Moreover, I cannot understand why the values from samplings after 72 hours were used, as the whole experiment took much longer. What was the RWC at the end?
7. That was a very short experiment. This kind of experiment may have value, but there can easily occur some random effects. Therefore, the experiments must be repeated (optimally several times) to confirm genotype-related effects. It is not clear whether the study was repeated.

Minor issues:

1. 210: In a sentence “After that, the samples were placed in a paper bag and kept in an oven at 65°C for 48 hours to record dry weight [30, 66, 67]” it is ridiculous to add references to support the drying in the oven. To add a reference to RWC method, references 67 and 68 are pretty enough.
2. In the introduction, the photosynthesis-related effects should also be emphasized. I suggest mentioning some of the influential studies on wheat, such as Photosynth Res 117:529-546, or some others. In connection to wheat genetic resources, another photosynthetic work is highly relevant:  Photosynth Res 136:245-255.
3. When discussing the water retention and water uptake, the residual (or cuticular) transpiration should also be considered and mentioned, as shown in a recent paper comparing wheat and wheat ancestors (Agronomy, 2021, 11 (3), 522). Although the authors did not assess this trait, it can be relevant and should be mentioned.
4. The sentence “Regardless of this, less attention has been given to early-stage drought screening” is not true – there are much more studies on seedlings than the serious field-based works. I suggest removing this sentence.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Belay et al studied a diverse wheat panel consisting of 43 genotypes that were treated with PEG-induced osmotic stress.  The panel included 40 diverse Ethiopian bread and durum wheat genotypes along with three Chinese bread wheat cultivars that were either drought tolerant (G33 & G34) or susceptible (G35). A total of 16 various physio-morphological and biochemical traits were characterized. The study generated an ample amount of data and were analyzed carefully using different methods. I have the following comments:

The authors should be careful in interpreting PEG applied osmotic stress as “drought stress”.  In many cases, the results obtained by PEG stress may not be directly translated to results obtained by soil drought stress. Even current study results implied that. G33 and G34 are standard Chinese dryland cultivars and have strong drought tolerance as stated in the abstract, and G35 is a wetland cultivar and supposed to be drought susceptible. However, in the current study, the authors found all of them had STI (stress tolerance index) less than 0.82 and were relatively stress sensitive, based on the PEG stress screening results. There may be a chance that both G33 and G34 are relatively less drought tolerant compared to over 20 other wheat genotypes. However, G34 has a STI of 0.17, which is very low, even lower than G35, which has a STI of 0.27.  

For this study, to make the results more reliable and applicable, I would suggest comparing the soil drought stress tolerance of several top selected drought tolerant and sensitive genotypes. For example, compare the soil drought tolerance between G12 (STI 4.28) and G25 (STI 2.37) with the two Chinese dryland cultivar G33 and G34, as well as with G35 and a couple of other sensitive genotypes selected based on STI.  

In addition, I have concerns about the small volume of PEG solutions used in this study. Ten seedlings were placed in one bottle containing 50 ml of solutions, which was a very small volume even that the solution was changed every five days.  Judging from Figure 1A, control plants generally lost 5-10 g of water per plant in two weeks, that’s 50-100 g of water for 10 plants in two weeks and about 18-36 g of water every 5 days in each bottle (10 plants).  This speed of water loss probably had concentrated the Hoagland solution, affected the nutrient concentration, and potentially caused osmotic stress in the control plants.  Because of different genotypes lost water in different speeds, the solution concentrating effect caused by gradual water loss was different for each genotype, which needs to be considered.

Minor comments: 

a large number of abbreviations are used in the manuscript and some of them appear to be less intuitive and conventional. I would suggest making some of the abbreviations slightly longer, e.g. abbreviate shoot as “St” rather than “S”, root as “Rt” rather than “R”, chlorophyll as “Chl” rather than “C”.

The discussion is a bit too long and can be more concise.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors made appropriate revisions or provided valid arguments that convinced me sufficiently.  The authors improved the manuscript significantly. The quality of the manuscript and the content make it interesting for the readers. I suggest accepting the manuscript in its present form.