Semi-Quantification of Lectins in Rice (Oryza sativa L.) Genotypes via Hemagglutination

Round 1

Reviewer 1 Report

The research on Semi-quantification of lectins in rice (Oryza sativa L.) geno-2 types via hemagglutination sounds promising. However, I have made some comments that need to be addressed. I think, more information is required from literature to support the findings of this experiment. Discussion should follow the order of the results. It seems out of order which makes difficult for readers to follow. please find my comments in the attachment as below 

Comments for author File: Comments.pdf

Author Response

Reviewer 1 The research on Semi-quantification of lectins in rice (Oryza sativa L.) geno-2 types via hemagglutination sounds promising. However, I have made some comments that need to be addressed. I think, more information is required from literature to support the findings of this experiment. Discussion should follow the order of the results. It seems out of order which makes difficult for readers to follow. Answer: Thank you for your worthy review. Discussion has been edited. please find my comments in the attachment as below: Line 46: Species name should be italic. Answer: Edited as suggested throughout the paper. Line 106: What is the purpose of including transgenic seeds and two local varieties in this study? Specify the need of additional samples for HA analysis. How many transgenic seeds are taken? Answer: We took 5 (five) types of transgenic seeds and two cultivated varieties (boti and fakhre malakand) for the comparison with their wild relatives. Line140: How many samples were tested for blood affinity for rice lectins. Please mention the total number of samples that were considered for this test. Answer: Total accessions taken were 217 (table 1) (the HA table 3 of accessions was missing by mistake which has been included in the manuscript) and 106 accessions (table 4) for further titre and specific activity evaluation. The data is explicitly presented in the Table 1. How did you make the observation, e.g. through naked eye? Answer: Yes sir, through naked eye. What is the parameter for comparisons? Please clarify how did you compare the blood affinity towards rice lectins. Answer: The literature about the interpretation of hemagglutination results is available. However, these observations and interpretations can vary from lab to lab, expert to expert, type and concentration of RBC’s, incubation time, nature of the sample agglutinin and method of hemagglutination assay. Also, there is no digital record of the microplate or titer determinations so the initial interpretation by experts is tiresome and commonly done in replicates. The range of potential variables and differences between expert readers make the comparison of inter-laboratory results difficult (Wood et al., 2012). The explanation is added in the article. Line 189-190: You need to describe Table 5, Figures 3 and Figure 4 in detail. What this table and figure explains for instance high or low HA etc…. Answer: Thank you sir! These have been removed to avoid confusion and their data merged in table 3 and 4. Line 192: you have used rabbit RBC for HA analysis, which has been described in HA analysis section (Line 116-130). However, in table 5, you have mentioned 5% broiler RBCs. Is it Broiler RBC or rabbit RBC in the title of table 5? Answer: Thank you sir! We have used the four types of blood for the comparative analysis to check which one is giving us better agglutination result. We have chosen Californian bred rabbit blood for all the samples. Furthermore, we have removed table 5 to avoid confusion. Line 211-212. It seems the number of samples for blood affinity for rice lectin test is variable. Please make it clear for reader how many samples/ rice accessions were tested for this test. Answer: Thanks! We have included the number i.e., 217 of rice genotypes in the mentioned line. As an important table 3 was missing from the first draft. Line 215: Table 5 does not exist in the manuscript. Answer: Acknowledged and rectified accordingly. Line 222: please check the references. Is it D.H Heard or Heard? Is it reference 38 or 39? Also check reference 39 and 40. Citation number in the test does not corresponds to the reference numbers. Please carefully check the citation in the whole text and make corrections. Answer: Thank you for the worthy suggestion. We have reset all the references and its numbering in the whole manuscript. Line 227: figure 11 and 12 do not exists in the manuscript. Answer: Thank you for pointing out. The figures have been removed to avoid confusion Line 237: in 163-165, you have mentioned samples were kept at around 45oC for rapid and accurate results. However, in the discussion you have mentioned 25oC. Which one is correct? Answer: The higher temp i.e., 45˚C is only used in case of low room temp, for instance in cold weather, otherwise the reaction is carried out at 25˚C/ room temp. Line 269: is Future prospects a different heading or a part of conclusion. Please clarify. Prospects would be based on your findings and how this finding will be appliable for future analysis. Those paragraph does not make sense to me. Answer: Thank you so much. Conclusion was written again with the related aspects.

Reviewer 2 Report

The authors describe the rice-derived lectins using agglutination assay. Rice lectins are helpful for environmental stresses including infectious agents and temperature. The rice lectin sources are from local rice accessions of IABGR/NARC Islamabad, Pakistan. HA has been applied for rabbit erythrocytes.  From 210 accessions, 106 showed the highest HA activity and the genotypes were analyzed for titer and specificity with human, chicken,  rabbits, and Californian-bred rabbits.  The classification of the genotype-based rice lectins are partially done and the basic results can be implicated for future studies on the biological activity against infections agents or etc. However, the present manuscript is just screening or analytic approach of rice lectins.

For further processing, the rices should be compared to the gentypes, discussing why such different HA acitivities are obtained from each different rice source. Unfortunately the present data just write the HA activities, without any meaningful significance. In addition, the rice lectins even as crude forms should be elctrophoretically separated and compared to each rice form.

The present MS can be recommended for substatial revision before accept

Author Response

Reviewer 2:

The authors describe the rice-derived lectins using agglutination assay. Rice lectins are helpful for environmental stresses including infectious agents and temperature. The rice lectin sources are from local rice accessions of IABGR/NARC Islamabad, Pakistan. HA has been applied for rabbit erythrocytes.  From 210 accessions, 106 showed the highest HA activity and the genotypes were analyzed for titer and specificity with human, chicken,  rabbits, and Californian-bred rabbits.  The classification of the genotype-based rice lectins are partially done and the basic results can be implicated for future studies on the biological activity against infections agents or etc. However, the present manuscript is just screening or analytic approach of rice lectins.

Answer: Due to large number of samples the study was confined to semi quantification of lectins via HA, titer and specific activity. Also we have compared different types of RBC’s affinity for rice lectins. Our future project is on its detailed characterization.

For further processing, the rices should be compared to the gentypes, discussing why such different HA acitivities are obtained from each different rice source.

Answer: We have answered this query in the discussion of the article and are thankful for highlighting this aspect.

Unfortunately the present data just write the HA activities, without any meaningful significance.

Answer: Added a meaningful description.

In addition, the rice lectins even as crude forms should be elctrophoretically separated and compared to each rice form.

Answer: Dear Sir, Your kind and precious comments are highly acknowledged. Our future project is the electrophoretic studies of these genotypes for lectins which is in progress. We have also done the genetic diversity of some of these genotypes in the recent study based on total seed storage proteins in (Gulzar et al., 2021). The remaining seeds will be analyzed for the electrophoresis process in our next project.

Round 2

Reviewer 1 Report

Hello Authors,

Thanks for improving your manuscript. Still, I have made few comments on the manuscript. 

Comments:

Line 111: Total number of accessions does not correspond to the source of origin. For instance, 4 accession from China, 177 from Pakistan, 19 from IABGR and 12 from IRIR = 212. But the total number of accession was 210. Please check it.

Line 167: Is there a full stop before out of 217. Please check

Line168: please remove , before 1. It should be 01 cultivated

Line 234: There must be a Figure number like 1, 2 or 3. That figure number may be a combination of few figures like Fig.1 (i-vii). I also wonder there is no explanation of figure (i-vii) in the text of the results section. Straightaway discussed for Figure viii. I also wonder what it represents A and B in figure i-vii. Please clarify these issues and write a paragraph about Fig i-vii in the results section.

Line 312: Please table 1 from the line. No need to repeat it again and over again.

Reviewer 2 Report

The authors have improved the manuscript appropriately and continually studying the rice species with the HA phenotype and genotype.

Thus, the present study should be accepted.