Standardization of an In Vitro Seed Germination Protocol Compared to Acid Scarification and Cold Stratification Methods for Different Raspberry Genotypes

Round 1

Reviewer 1 Report

Overall, this experiment only focused on the effect of treatment on germination. The design of the experiment is relatively simple, and data shown in MS are less.

 

1.         Some experiments can be supplemented appropriately to extend the results.

 

2.         Does germination treatment have a better effect on growth?

 

3.         It was suggested that the part of “Introduction” should be revised into several separate paragraphs.

 

4.         In particular, the Introduction needs much more detail.

 

5.         Line143: “aT1, bT3, cT5, aT2, bT4, cT6,” The specific treatment methods of the six methods are detailed in the Materials and Methods section. Perhaps changing to bT1, bT2, cT1, cT2 is more convenient for readers to identify.

 

6.         Line 155: “c) longitudinal incision,” can it be understood as cutting only one wound, rather than halving the seeds as in b) transverse cutting.

 

7.         Line 189: “AB20,22-AB20,09-AB20,17” The origin of hybrid varieties (male parent and female parent) should be explained in a table.

 

8.         Does this germination protocol have a good guiding significance for other species?

Author Response

1. Some experiments can be supplemented appropriately to extend the results.

Dear reviewer, thank you for reading our manuscript and for your useful comments.

Additional trials are ongoing on other raspberry genotypes to evaluate their efficiency of germination after in vitro treatment. In addition, phenotypical characterization in field condition of the germinated plants described in this study is in progress. However, these additional data would require several months of analysis before to be considered for a publication. They will be probably presented in a future manuscript.

We hope that the data reported in this manuscript, obtained through several repetition and independent experiments, on several different genotypes, could be enough to demonstrate the efficiency of the protocol optimized and described, which is the main aim of this study.

2.         Does germination treatment have a better effect on growth? 

Dear reviewer, we have not observed any effects on plant growth after treatment, neither positive nor negative. The advantage observed by applying the in vitro germination protocol, is the shortest time necessary to germinate the seeds, and the highest efficiency of germination regardless the genotype, compared to not treated/scarified seeds.

In addition, as mentioned above, phenotypical characterization of the germinated plants, from both in vitro germination and chemical scarification, are now under study in field condition. From our preliminary observations, no differences have been observed among all the plants obtained, regardless of the applied treatment. Differences observed by now seem only to be linked to the genotype itself.

3. It was suggested that the part of “Introduction” should be revised into several separate paragraphs.

Dear reviewer, we agree with your comment. We revised the introduction and separated it into different paragraphs.

4. In particular, the Introduction needs much more detail.

Dear reviewer, we revised the introduction following your suggestion. In particular from line 60 up to line 118  we included additional information.

5. Line143: “aT1, bT3, cT5, aT2, bT4, cT6,” The specific treatment methods of the six methods are detailed in the Materials and Methods section. Perhaps changing to bT1, bT2, cT1, cT2 is more convenient for readers to identify.

Thank you for this consideration. We modified the nomenclature related to the different treatments as follows:

 

Treatment	Previous nomenclature	New nomenclature
Uncut seed 24°C	aT1	aT2
Uncut seed 4°C->24°C	aT2	aT1
Transverse cut 24°C	bT3	bT2
Transverse cut 4°C->24°C	bT4	bT1
Longitudinal incision 24°C	cT5	cT2
Longitudinal incision 4°C->24°C	cT6	cT1

In addition we also included these corrections in figure 1.

6. Line 155: “c) longitudinal incision,” can it be understood as cutting only one wound, rather than halving the seeds as in b) transverse cutting.

Yes, the term “longitudinal incision” refers to an engraving of the seed, without cutting it in two halves. We better clarified these treatments at lines 176-177.

7. Line 189: “AB20,22-AB20,09-AB20,17” The origin of hybrid varieties (male parent and female parent) should be explained in a table.

We thank the referee for this comment but as indicated in the acknowledgments this work is part of a breeding program carried out for a company that asks us to keep the crossbreed combinations confidential. For this reason, we cannot include them. In any case, in our opinion this information can be considered of minor importance for this manuscript as it is mainly aimed at describing a new, more efficient method of seed germination that can be very useful for breeders working on raspberry and other species.

8. Does this germination protocol have a good guiding significance for other species?

Thank you for this relevant question. As mentioned above, the in vitro germination protocol optimized in this study can be very useful also when applied to other species, if properly optimized case by case. We included some information in the discussion section related to similar approaches already applied to other fruit tree species characterized by the same low percentage of germination as raspberry, specifically from line 364 up to line 374.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The present study describes developing a new protocol to efficiently overcome raspberry seed dormancy. The more efficient method has been applied to 14 cross-combinations between two main cultivars. The optimized protocol presented in the manuscript resulted in a significant increase in the germination rate of seeds when compared to the standard protocol.

The Introduction section is well-written and provides enough feedback and logic for the purpose of the study.

The Method section is sufficiently detailed.

There is a clear presentation of the results in the paper, and the figures and figure legends are well explained. Below are a few comments I have.

- What was the reason for choosing Polka and Tulamagic cultivars for the experiment and cross combination? Do they have commercial importance? Were they selected because previous methods are optimized for these cultivars? Would you be able to add the reason to the manuscript?

- In the Result section, Part 3.1, lines 182-185 could be part of the Methods rather than Results.

- In the Discussion section, lines 293 to 295: The sentence starting with “The poor germination” is not clear and needs re-wording.

- In the Discussion section, lines 283 to 302: the information introduced in the Introduction section is repeated here. Considering that this part does not discuss or interpret the results, it can be reduced.

 

- In case the manuscript is not a short communication, the Discussion section might benefit from reviewing more previous studies and literature. Previous studies may not have directly addressed dormancy/germination protocols in this species or subspecies. However, they can help develop the concepts and discuss dormancy, low germination rate, and treatments.

Author Response

- The present study describes developing a new protocol to efficiently overcome raspberry seed dormancy. The more efficient method has been applied to 14 cross-combinations between two main cultivars. The optimized protocol presented in the manuscript resulted in a significant increase in the germination rate of seeds when compared to the standard protocol.

The Introduction section is well-written and provides enough feedback and logic for the purpose of the study.

Thank you for reading our manuscript and for the useful suggestions.

-The Method section is sufficiently detailed.

-There is a clear presentation of the results in the paper, and the figures and figure legends are well explained. Below are a few comments I have.

- What was the reason for choosing Polka and Tulamagic cultivars for the experiment and cross combination? Do they have commercial importance? Were they selected because previous methods are optimized for these cultivars? Would you be able to add the reason to the manuscript?

We thank the reviewer for this comment. We included some information about the two cultivars studied and reasons why they were selected to start our investigations from line 107 up to line 114: "The efficiency of this protocol was first demonstrated on seeds from open pollination of two raspberry genotypes, “Polka”, a primocane fruiting cultivar, and “Tulamagic”, a floricane fruiting cultivar. Both these genotypes have been included in several breeding programs mainly aimed at improving the quality of raspberry fruit [30,31]. These two cultivars can be considered representative for several raspberry genotypes, due to their different fruiting cycles and their importance for the breeding of this crop. For these reasons, they were chosen as the two starting genotypes to carry out the experimental trials described in this study."

- In the Result section, Part 3.1, lines 182-185 could be part of the Methods rather than Results.

Thank you for this suggestion. We decided to remove these sentences, because scarification and stratification steps are already detailed in the materials and methods section, and later summarized graphically in figure 7.

- In the Discussion section, lines 293 to 295: The sentence starting with “The poor germination” is not clear and needs re-wording.

Dear reviewer, we rephrased this sentence in the manuscript as follows:

The low germination efficiency observed, coupled with the long time required for stratification, (i.e., 3 months), make this procedure lengthy and inefficient.

- In the Discussion section, lines 283 to 302: the information introduced in the Introduction section is repeated here. Considering that this part does not discuss or interpret the results, it can be reduced.

We thank the referee for this comment. We reduced this part in the discussion section.

- In case the manuscript is not a short communication, the Discussion section might benefit from reviewing more previous studies and literature. Previous studies may not have directly addressed dormancy/germination protocols in this species or subspecies. However, they can help develop the concepts and discuss dormancy, low germination rate, and treatments.

Thank you for this suggestion. We included more information in the discussion section related to other studies carried out in this and other species from line 364 up to line 374.

In addition, we better discussed the concepts of dormancy, low germination rate, and treatments related to raspberry and other plant species also in the introduction from line 60 up to line 88.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors developed a protocol of optimization of raspberry seed germination, which was demonstrated to show high germination efficiency for several other cross combinations independent of their parental genotypes. Overall, the manuscript is well written, however, there are several points that I would like the authors to pay attention to improve this manuscript.

1. Fig 5. How many seeds were examined for each genotype?

2. Please discuss the why transverse and longitudinal cut show difference on the seed germination?

3. It would be nice if a more detailed protocol can be provided.

Author Response

The authors developed a protocol of optimization of raspberry seed germination, which was demonstrated to show high germination efficiency for several other cross combinations independent of their parental genotypes. Overall, the manuscript is well written, however, there are several points that I would like the authors to pay attention to improve this manuscript.

 

1. Fig 5. How many seeds were examined for each genotype?

We thank the reviewer for reading our manuscript and for the useful comments.

In figure 5 the number of seeds used is 125 for each condition applied and for each genotype. As reported in the materials and methods section 2.3, ten petri Plates were prepared, each containing 25 seeds for each genotype and type of mechanical treatment, half of them were placed directly at 24 °C, and the second half at 4°C for two weeks and then moved to 24°C, as for the first group. We better specified at line 192 that, in addition to data on seed germination percentage for each type of method, also the time of germination was acquired, as showed in Figure 5. We also included the total n. of seeds used for each condition in the legend of figure 5. 

2. Please discuss the why transverse and longitudinal cut show difference on the seed germination?

Thank you for this comment. We included a possible explanation on the reason why we observed differences between the two mechanical treatments in the discussion section at lines 349-356, also considering other results from the literature: "...the longitudinal incision of the seed (treatment “c”) was less effective that the cut in half of the seed in terms of germination efficiency. This result could be related to the ineffective lesion of the raspberry seed endocarp, which, on the contrary is completely broken through treatment “b” (cutting the seed in two halves), leading to the highest observed seed germination rate (Figure 4). Similar results were shown in another study, where raspberry seeds with nicked endocarps failed to germinate until the testa and endosperm were injured and exposed to the external environment [16].

 

3. It would be nice if a more detailed protocol can be provided.

Dear reviewer, we included some more details in the materials and methods section.

Author Response File: Author Response.pdf

Reviewer 4 Report

In this manuscript, Pergolotti et al. develop an optimized protocol to substantially increase the germination efficiency for all raspberry genotypes. They found that the in vitro seed mechanical treatment, especially transverse cutting, is an efficient way to promote the germination rate, no matter the temperature applied. Overall, the result is interesting; however, I have some concerns and suggestions.

1.    I do not understand how the standard deviations in Figures are made. The typical deviation is not from the mean?

 

2.    In this manuscript, the author performed many treatments and made a comparison of the gemination efficiency. They used many short abbreviations for each treatment but without clear definitions. This is really confusing me because I always forget what “bT3” or “bT4” stands for. Thus, I suggest the authors make a table to illustrate all the treatments in this study and give abbreviation names after them. 

Author Response

1. I do not understand how the standard deviations in Figures are made. The typical deviation is not from the mean?

We thank the referee for noticing this mistake. Standard deviation values were wrongly inserted in the graphs. We confirm that Standard deviation was calculated from the means of three independent experiments, we corrected it in figures 2, 4, and 6.

 

2. In this manuscript, the author performed many treatments and made a comparison of the gemination efficiency. They used many short abbreviations for each treatment but without clear definitions. This is really confusing me because I always forget what “bT3” or “bT4” stands for. Thus, I suggest the authors make a table to illustrate all the treatments in this study and give abbreviation names after them. 

Dear reviewer we agree with your comment. We simplified the nomenclature for treatments as follows, reporting these indications also in figure 1.

We decided to not include also a table with the same information, because it could seem redundant with figure 1.

Treatment	Previous nomenclature	New nomenclature
Uncut seed 24°C	aT1	aT2
Uncut seed 4°C->24°C	aT2	aT1
Transverse cut 24°C	bT3	bT2
Transverse cut 4°C->24°C	bT4	bT1
Longitudinal incision 24°C	cT5	cT2
Longitudinal incision 4°C->24°C	cT6	cT1

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

It is a great pity that the authors are unwilling or unable to address my concerns sufficiently. In particular in this second round for  comments 1,2,7,8. 

Author Response

It is a great pity that the authors are unwilling or unable to address my concerns sufficiently. In particular in this second round for comments 1,2,7,8. 

 

1. Some experiments can be supplemented appropriately to extend the results.

 

Dear reviewer, sorry if we were unable to address your concern, but in our opinion, it is not clear which additional experiments you suggest to include in the manuscript.

We assume that you are referring to the evaluation of vegetative and qualitative parameters on the germinated plants obtained through the two germination protocols described. With this hypothesis, we included in the results section additional photos in Figure 3, showing that plants germinated with the two protocols showed quite a homogeneous behaviour after germination (see new figure 3). We agree on your idea that seeds of the same population can have a different variability depending to the germination procedure, if only because in vitro germination favours the germination of seeds that cannot germinate with the in vivo protocol. However, we think that it is not so easy to estimate this possible different variability and in short time. Indeed, the germinated seeds obtained by applying the two protocols described in this study (in vitro germination and chemical scarification/stratification), have different timing of germination, as described in the manuscript, and were transferred in the field in different moments.

Therefore, we do not consider reliable the evaluation of the different population at this early stage. However, if it can be considered useful, we propose to add an additional photo of the different plants in the field (Supplementary figure 1). For sure we will take in consideration this comment and in the field evaluations, that will continue in the next following seasons, we will analyse the variability of the seedlings obtained with the different germination methods.

We reiterate that the aim of this work is to describe the different efficiencies of the proposed germination methods and not to analyse the variability generated in the different populations of seedlings. This will be the objective of subsequent work which, in order to have scientific value, must be carried out in the field and will require at least 2 years of evaluation. Such considerations have been included also in the discussion.

 

In particular, we added at line 410 the following paragraph: “Seedling populations obtained from the different cross combinations with the two germination protocols were easily developed in greenhouse, showing similar level of development and then were planted in the field (Supplementary figure 1). At this stage, it was not possible to assess the potential effect of the seed germination method on the genetic or phenotypic variability of the seedling populations. This because the germinated seeds obtained by applying the two protocols described (in vitro germination and chemical scarification/stratification), had different timing of germination, and were transferred to the field in different moments.

The variability generated in the different populations of seedlings obtained with the different germination methods will be the objective of subsequent work which, in order to have scientific value, must be carried out in the field and will require the pheno-morphological assessment of all seedlings for at least two cultivation cycles.”

 

We hope that the described experiments, and the related results presented in this study, and the additional comments now included, are enough to show the efficiency of the in vitro protocol we optimized. 

 

2. Does germination treatment have a better effect on growth? 

 

Dear reviewer, the in vitro germination treatment apparently had no better or worse effect on the growth of the seedlings. We added this information in the results section at line 249:

 

“This kind of treatment did not negatively affect the development of the germinated seedlings, which in short elongated and rooted in vitro, and after were easily acclimatized to in vivo conditions under greenhouse (Figure 3c-f).”

 

We also added some additional photos in figure 3, which show in vitro elongation and rooting. We also included in figure 3, two photos representing the acclimatization process and the final in vivo establishment of the in vitro germinated plants, which show their homogeneous and normal development and growth. In order to have an objective comparison of vegetative and qualitative parameters of these plants compared to those obtained by the standard scarification and stratification method, we will collect data during the coming cultivation seasons, as mentioned above.

 

7. Line 189: “AB20,22-AB20,09-AB20,17” The origin of hybrid varieties (male parent and female parent) should be explained in a table. 

 

Dear reviewer, we are sorry to not to be able to address to this comment, but as already reported in the first round of revision, this work is part of a breeding program carried out for a company that asks us to keep the crossbreed combinations confidential. For this reason we cannot fulfil your request. However, we reiterate that this work aims to compare the germination efficiency of two seed germination methods, so as to facilitate the work of breeders in the delicate phase of seed germination. The assessment of the variability generated by the different crossing combinations and possibly also by the seed germination method will be the objective of the work that will continue in the coming years.

 

8. Does this germination protocol have a good guiding significance for other species?

 

Dear reviewer, we are sorry to not to have addressed sufficiently this comment. In addition to what already added, we have now included in the introduction section the additional following paragraph from line 114 up to line 119:

“In a second phase, by applying the optimized protocol on seeds from 14 different cross combinations, it was demonstrated that the in vitro method remains the more efficient also when applied to seeds originated by different cross combinations, therefore, resulting as new seed germination technique helpful to maximise seed germination and speeding up breeding programs in Rubus spp. and other crops with similar problems in low seed germination.” 

 

While, in the discussion section we added the following paragraph from line 376 up to 389:

 

“The in vitro germination protocol described in this study represents a useful guideline for the development and optimization of similar methods to be applied for other plant species, characterized by seed dormancy and low germination rates, as already reported in literature. For instance, in strawberry (Fragaria x ananassa) it was described an in vitro protocol increasing the efficiency of germination and shorten germination time, when achenes were cut across the embryo axis and were placed in contact with the culture medium [35]. In another study aimed at increasing seed germination of Ilex dumosa R., transversal cutting of sterilized pyrenes and their subsequent in vitro culture, induced a maximum germination rate of around 70% after two months of in vitro culture, compared to the whole pyrenes, which reached a maximum germinability of 37% after eight months after sowing [28].

Therefore, the in vitro seed germination protocol described in this work can contribute to speed up breeding program in Rubus spp. and in all other plant species having low seed germination rate.”

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

The author's reply has solved most of the problems.