Somatic Embryogenesis and Flow Cytometric Assessment of Nuclear Genetic Stability for Sansevieria spp.: An Approach for In Vitro Regeneration of Ornamental Plants

Round 1

Reviewer 1 Report

The article is a valuable contribution for micropropagation (somatic embryogenesis) of ornamental Sansevieria species. However, it has some parts that should be better desighes, in particular regarding flow cytometry measurements.

Title: Somatic embryogenesis and ploidy assessment for Sansevieria 2
spp.: an approach for in vitro regeneration of ornamental plants. Comment: no ploidy assessment was done.

Manuscript:  Do not use hyphenation particularly not in latin names.

Tissue culture protocols

No major corrections are needed but discussion regarding explant response and nuclear DNA variation should be based on reliable data. Some discussion regarding well known disrupting action of 2,4-D and TDZ?

Regarding flow cytometry using DAPI is valuable in order to define variation from donor (mother) material and regenerants (by tha way for us LB01 in combination with DAPI gave non optimal response compared to DAPI in Otto buffers)  but its relative nuclear content should be given in units like picograms. Cited references using PI - Šmarda 2014 and Zonnevelt 2005 differ (1.24 or 2.60 respectivelly). Šmarda concluded that sample was diploid, while Zonnevelt make no ploidy estimation. Species were examined for ploidy by chromosome counting by Nazeer and Khoshoo 1984 and an earlier paper.  Cytology of Some Species of Sansevieria Thunb. Cytologia 49: 325-332, 1984 (measured were S. concinna, S. parva, S. pearsoni and S. suffruticosa)  and the first showed 120 chromosome and the other three 40. So probably 40 represents 2n = 2x value (also possible 4x value).

Flow cytometry

Flow measurements were done in an unusual way using 2.5 μ m polystyrene microspheres (stained?). These very small beads (should be at least 10 or 30 um) are normally used to optimise flow measurements but not as internal standard. Regarding its already measured genome size (above) a species around 2 pg should be used (like Trifolium repens or similar). Since beads were displayed on the very left side of panels, mistakes are very likely. All histograms are shown not as actually obtained but as estimated using software application which give unrealistic low CV value (by the way no original CV values are given).  For these reasons results are doubtful. Also Table one gives no nuclear DNA values.

I suggest that nuclear DNA measurement should be repeated using another procedure. The most interesting finding might be measurement of regenerant from style of S. parva. Haploid?

Author Response

see file in attach

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript represents the results of a broad and interesting study and would be of great value for the respective audience. Therefore I recommend it to be published in the Horticulturae Journal

Some minor addressing of typos is needed, only several examples follow below:

- “in vitro” should be in italics in the title.

- Line 40: “drier part of eastern and southern Africa” should be “drier parts of eastern and southern Africa”

- Line 58: “organogenesis from leaves explants” should be “organogenesis from leaf explants”

- Line 89: “accessions name are reported” should be accessions names are reported

- Line 108: “Netherland” should be “The Netherlands”

- Line 122: “culture condition” has to be “culture conditions”

Then major concerns regarding the scientific content need to be addressed:

In Material and methods “2.1. Plant Material”  specify more concretely how many explant types were excised: stigmas/styles, anther/filament, ovary - were they separated into five different explant types or were they three type of explants. Please, shortly comment on the haploid (n) or diploid (2n) character of the different explants. Currently explants are described in Lines 93 and 98 with slight differences. It has been stated that they are non-fertilized, but still it would be useful to concretely describe and abbreviate the explant types inoculated into the media and comment on their (n) or (2n) number at this point of description of Material and Methods. In Fig. 2A, cited here, four explant types are illustrated, but they are not named concretely in the Figure legend. I would suggest giving abbreviations of the different explant types and further merging these abbreviations with the ones of the media on which the respective growth was induced, so that the obtained lines could be clearly denoted throughout the following studies.

The paragraph in Lines 111 - 117 needs to be more clearly explained. Was it that explants from the mother plant (has to be already made clear how many explant types) were initially placed in the T4, T5 and T16 media and transferred into the same media composition at 60 d intervals (several times, how many?)? Were the lines obtained from the different combinations (explant type/media type) kept as initially inoculated for these successive subcultures at every 60 days? Then it is described that  explants showing embryonic response (while on T4, T5 and T16 media? So it is necessary to state which explants from which of the media) were transferred to  basal MS-medium deprived of PGRs and kept for one month. So, the origin of the “Embryogenic calli” described in Line 114,  transferred to Petri dishes containing 20 ml of basal solid MS medium is not very understandable. Was it obtained after one month on PGR-free medium?

2.3. Embryo germination, plant development and acclimatization - after addressing the content above, the origin of the “Individual somatic embryos” in Line 119 has to be now clear. Now it is not clear where this callus and embryos (Line 119) came from, because in the above paragraph several consecutive steps of transfer through media lacking PGR are now described.

Line 124: Please describe more clearly here what occurred between embryo germination (as stated “when there was root extension”) and washing and transfer of the plantlets to soil.

Line 126: Regarding cultivation of the plants in soil, it is not clear which are the culture condition “as described above”. It is already stated in this paragraph (Line 126) that “The potted plants, covered with transparent polyethylene bags to maintain temperature and high humidity (Fig. 2G), were placed in a climate chamber at 25 ±1 °C”. If there is something to add (light intensity or other), please just write it concretely, because all other conditions above concern in vitro plants and confusion is being generated. Since “high humidity” (Line 127) and “reduction of humidity” (Line 129) are mentioned, please describe how this was done (by puncturing the polyethylene bags), only if it is available, some values of humidity (high, reduced) could be indicated.

2.4. Flow Cytometry and Ploidy evaluation

The information of the obtained lines (illustrated in table 1) have to be illustrated by the abbreviation discussed above - it has to be clear which of the explants of the mother plant on which medium yielded these regenerants. The understanding of the diploid or haploid nature of the tissues would be very useful to relate to these results.

Addressing of all these remarks from above would significantly improve the interpretation of data and support the statements made in the “Results and Discussion” section. Just one example of how this addressing is needed with the opening of the section is given below:

3. Results

3.1. Plant regeneration

Line 174: “Callus formation was observed after 60 days from the beginning of the culture under all the PGR combinations tested. Regeneration was obtained from all genotypes with differences according to explant type and medium used”. This statement needs to be illustrated by concrete examples following a combined medium/explant type abbreviation. Otherwise such statements have to be specified by “data not shown” which is obviously not the case. Thus, for example - it has to be clear for S. concinna - from which explant on which medium did “S-CNR-014/1” originate, and so on.

Please, try to revise the manuscript to bring more clarity.

Author Response

see file in attach

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I believe that draft manuscript was now improved and can be accepted as such.

Reviewer 2 Report

The authors have considerably improved the manuscript according to the remarks of the review so it can now be accepted for publication.