Efficient Procedure for Induction Somatic Embryogenesis in Holm Oak: Roles of Explant Type, Auxin Type, and Exposure Duration to Auxin

Round 1

Reviewer 1 Report

The manuscript describes an experiment to optimize induction of holm oak embryogenic cultures from non-zygotic explants, enabling cloning of known genotypes, by testing explant types, auxin types, concentrations and exposure durations. The results identified a new induction protocol that substantially raised induction frequencies over existing protocols, but also showed that optimal auxin treatments and explant types varied with genotype. Overall, I think the manuscript is a useful contribution to the field, particularly because oaks are a high-value genus that has been challenging to clonally propagate, especially from mature tree tissues. While I do have a few issues with descriptions of statistical analysis results and extrapolation of the results to application for mass clonal propagation , most of my comments concern suggested changes to the writing, which is good for the most part.

Specific comments

1.      Title and elsewhere.  The phrase “time exposure” is awkward.  I suggest substituting “exposure duration”

2.      Line 36. I am not sure what is meant by “intervenes in” here. Maybe “contributes to” would be more appropriate?

3.      Line 67. See comment #1

4.      Lines 74-75. I would rephrase this sentence, “…firstly producing dedifferentiation and lastly embryogenic differentiation” to read “… promoting dedifferentiation followed by embryogenic differentiation”

5.      Line 82. I rarely see this family called “Fagacea”.  Fagaceae is much more commonly used.

6.      Line 140. Change “added with” to supplemented with”

7.      Line 141.  I think you mean “NAA” rather than “ANA”.

8.      Lines 142-146. This is a very long sentence. Suggested re-wording: “The IAA and NAA treatments were chosen based on previous studies of SE induction in holm oak [20].  The IBA treatment was chosen following the appearance of somatic embryos when this treatment was used to induce adventitious root formation on E00 axillary shoots…”

9.      Line 149-150. Information needs to be added regarding the frequency of transfer of the explants during the 24 weeks they spent on basal medium. Also, “standard conditions” is vague. Do you mean the same temperature and dark conditions described above for the cultures on the PGR treatments?

10.  Line 179. You should mention here that interactions between the main factors were also investigated.

11.  Line 196. To avoid confusion, start this sentence with: “With the NAA treatment, calli appeared only around wounded areas…”

12.  Line 197.  I think you mean IAA instead of AIA here.

13.  Line  199.  Confusing. You were talking about the IAA treatment in the previous sentence, so what do you mean here by “in this IBA treatment”?

14.  Line 202.  Change “indirectly” to “indirect”

15.  Fig. 2. Photo of structure in Fig. 2D appears more like a bud than an embryo. Do you have a photo of a structure arising from a leaf explant that looks more like an embryo? Normally, some histology would be called for to confirm the structures have closed vascular systems, which would settle whether the structure is a somatic embryo or not, but the other structures in the figure look like embryos to me.

16.  Lines 241-250 and elsewhere.  While I understand that the treatment and interaction effects are challenging to describe in a 3-way factorial experiment, the authors need to be careful with statements describing the results. First, the identification of significant interactions, the like the PGR treatment by genotype interactions with shoot apex explants, may mean that the main treatment effects (treatment and genotype) are actually uninterpretable, despite the ANOVA identifying them as significant. Second, because of the significant interactions, running means comparisons (Duncan’s, Tukey’s tests) is probably not worthwhile. However, this also means that statements like “The induction rates were significantly higher with the shoot tips of Q3-SE cultured with NAA (33%) for two weeks” are not supported, since no means comparisons were performed, even though 33% is the highest value in the table.

17.  Lines 247-249. This sentence is not accurate. Q3-SE treated with IAA for 2 weeks actually had lower induction than the other two genotypes, although the rate with NAA treatment (33%) was higher.

18.  Lines 249-250. This sentence is not accurate. According to Table 1, 2 weeks of NAA treatment did induce SE in Q10-SE.

19.  Lines 268-269. This sentence needs rephrasing. I suggest something like: “Also similar to what was found with the shoot apex explants, three of the four possible interactions between treatments had significant effects on SE induction (genotype x auxin treatment, …”

20.  Lines 312-313 repeat the same sentence found in lines 306-307.

21.  Line 337.  “Remarkable results” is a strange subheading. Maybe “Overview of results”?

22.  Lines 434-435.  I would replace “rejuvenate” with “rejuvenating”

23.  Line 437. Should read: “…when axillary shoot cultures of Q3-SE and Q10-SE…”

24.  Line 454.  See comment #1

25.  Lines 517-518. Change “the nodes very young” to “very young nodes”.

26.  Line 528. Change “non-somatic embryo” to “no somatic embryo”

27.  Lines 548-549. Costs were not measured in this study and nothing was included about contamination, so I would delete this statement or modify it to make clear that these are possible or likely benefits.

28.  Lines 549-554.  This is a very long sentence. Also, I think readers need to be reminded, either here or in the Discussion section, that this study only concerns an advance in SE induction and that multiple bottlenecks remain to be overcome before SE could be employed for mass propagation or integration with breeding programs, including scaled-up SE production, high frequency germination and conversion, and improving somatic seedling quality. There is nothing in this manuscript about the relative productivities of the cultures derived from different explants or treatments via repetitive embryogenesis following the induction of the initial 1-3 somatic embryos per explant.

29.  Lines 552-553. Change “phenotype adults” to “adult phenotypes”.

Author Response

Please see file.

Reviewer 2 Report

In this manuscript titled, “Efficient procedure for Somatic Embryogenesis in Holm Oak: roles of Explant type, Auxin type, and Time exposure to auxin”, Martínez and Corredoira present on somatic embryo induction in this important oak species, using a few different media and explants. This work was well written and the information in the manuscript is noteworthy but the authors should correct some errors in the manuscript and open their minds about what they are seeing in their cultures and how they can expand their understanding of the overall process of somatic embryogenesis in plants. 

Please explain oak decline – is this a pathogen or general decline from environmental changes – this was not clear – include references that an English-speaking reader can access.

Cryopreservation does not lead to improving forest species – revise

Naphthylacetic is not the same as Naphthaleneacetic acid – you are using the latter

Indolebutyric acid is misspelled.

“close to the site of meiosis” should be removed – this is not an accepted measurement

Two treatments were auxin and cytokinin, while one was two auxins – the selection of these 3 different media was a little unusual and minimally explained. Please elaborate.

Please check to see if you are using 100 x 15 mm plates which are the standard in most places.

Please explain in more detail why this procedure is an improvement of the PCTOC paper. It seems like more embryos were obtained and much more detail was provided in that paper. It also seems like some of the media were exactly the same and more media were evaluated in that paper. This paper presents like more of a procedural paper with little new data.

The authors should comment on plant regeneration from these somatic embryos and state that they have, or have not been able to recover plants yet from the somatic embryos obtained in this study. 

Please seriously consider the following information on somatic embryogenesis – this reviewer has worked with embryogenesis for many years with many different plants and does not want to offend the authors. I am just trying to assist you and open up some additional possibilities for research.

As the authors are aware, 2,4-D is the primary auxin used to induce somatic embryogenesis and the weaker auxins are usually less effective. There are very few reports on using IAA to induce embryogenesis because it is broken down so quickly by plant cells, since it is the only hormone. Usually, as I think the authors are aware, the embryos induced in the presence of auxin, develop when the tissue is transferred to an auxin free medium. But, some auxin is retained by the tissue.

I think that the authors induce DEVELOPMENT of embryos on transfer to growth regulator free medium. I think that embryos and embryogenic tissue are produced prior. The authors need to learn how to recognize proliferative embryogenic callus. The images that the authors show in this and previous papers are very large developing embryos. But, these embryos are derived from smaller embryos and embryogenic tissue, and it takes a long time to see the embryos, because these are developing embryos and they grow so slowly under their conditions. Typically, embryos are induced and proliferate (continued induction) with auxin and develop in the absence of auxin. When embryogenic tissue is placed on media with intermediate levels of auxin, you will get some poor proliferation and some poor (and abnormal) development. When you separate induction and development, proliferating embryogenic callus will first form and this is difficult for some to recognize. It is interesting that proliferative embryogenic callus from many plants looks the same. Sometimes, you will have some developing embryos on this callus; sometimes not. I have seen many people obtain beautiful embryogenic callus and not know it, because they did not know what to look for. You may have embryogenic callus at the base of a whorl or group of embryos – look for this tissue to start. They did not show that here but may have obtained that in the PCTOC paper. When embryogenic callus is transferred from auxin-containing media to a growth regulator-free embryo development medium, the developing embryos will have more normal looking cotyledons and hypocotyls and may even germinate more efficiently. 

The authors should be commended on the patience that it took to recover their embryos but they need to think more about these ideas above and look to the literature to expand their understanding of the overall process.

Author Response

Please see file.

Author Response File: Author Response.pdf