Effect of Light Spectrum, Sucrose Concentration, and 6-Benzyl-aminopurine on In Vitro Adventitious Bulb Formation in Tulipa tarda

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author,

The manuscript presents interesting original results on the method of micropropagation of Tulipa tarda by direct regeneration of microbulbs on bulb scales, and the factor studied is both the composition of the medium and the effect of a particular light spectrum on organogenesis.
The paper is clearly written, the results presented clearly.
I believe that the work needs only a few minor corrections. 

1) Introduction could be improved a little bit. For example, the information about the low natural reproduction rate of tulips is very general, and giving a literature reference here 5 - which is a whole monograph of several hundred pages - is incorrect, the citation should be more precise.

2) The chapter Materials and Methods needs to be improved by adding information The chapter needs to be improved by adding information on whether the MS medium was supplemented with vitamins or not, whether inositol was added or not, and by adding information on whether the cultures were passaged onto fresh medium every 4 weeks or not. 

3) In my opinion, the legends under Tables 1-3 need to be corrected. In Tables 1 and 2, only significance was observed at p≤0.001, so information about the other significance levels is redundant. In Table 3, the significance of differences between the averages was observed at two levels significantly, so information about their three levels is unnecessary. In turn, the explanation of what “ns” means is missing.

Author Response

Dear reviewer,

I would like to thank you for sending me all the comments raised by you and the referees, which helped me to improve the manuscript.

I would like to explain as follows:

Comment 1: the Introduction have been improved – the 5^th Reference was clarified;

Comment 2: the Materials and Methods was improved about information concerning: vitamins, inositol, passages frequency;

Comment 3: captions under the tables have been corrected.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The article should not be accepted in its current form. It requires substantial revision to improve the methodology, statistical analysis, and discussion of results within the context of the most recent literature.

The title is too generic and does not highlight the key variables of the experiment, such as sucrose concentration and the presence of BAP. The term "determine" suggests a causal relationship that was not experimentally demonstrated. The study only evaluates the effects of different light conditions, without definitive evidence that light determines organogenesis. This is just a suggestion: "Effects of Light Spectrum, Sucrose Concentration, and BAP on in vitro Adventitious Bulb Formation in Tulipa tarda."

 

The abstract contains redundancies and lacks clarity. For instance: "The study demonstrates that organogenesis from bulb scales is an effective and efficient method for T. tarda propagation, providing a method for mass production and offering potential for commercial cultivation of this ornamental species." This sentence is redundant; it is already known that organogenesis is a technique used for propagation, so stating that it is effective does not add novelty. Additionally, "mass production" lacks context, as no comparison with commercial methods is provided to justify this claim.

 

"Despite the development of many protocols for the micropropagation of tulips, an efficient propagation method suitable for commercial production has not yet been established [10]." This statement is too strong without sufficient evidence. Several studies have already reported efficient tulip propagation methods. Reference [10] (Yasemin & Beruto, 2024) is not specifically about the absence of efficient commercial methods, and its use in this context is misleading.

 

"Ten replicates were exposed to each of the four light and four media combinations." The number of explants per treatment is not clearly specified. The reader does not know the total number of replicates. Additionally, there is no justification for using fluorescent light instead of LEDs, which are now more common in organogenesis studies.

 

There is no information on relative humidity and ventilation control, which are crucial factors for preventing hyperhydricity and abnormal growth in in vitro cultures.

 

It is not mentioned whether antioxidants were supplemented to prevent explant browning.

 

"Under these conditions, the largest number of adventitious bulbs was formed throughout the entire experimental period, and after 12 weeks of culture, there were 12 adventitious bulbs per explant." This number is not contextualized: how does it compare to other studies? The result may seem relevant in isolation, but statistical variability is not discussed.

 

There is no statistical assumption testing, such as data normality tests (Shapiro-Wilk or Kolmogorov-Smirnov) or homogeneity of variances tests (Levene or Bartlett).

 

In Table 1, the interaction between light spectrum and medium composition is mentioned as statistically significant, but there is no visual indication of which groups actually differ from each other. Letters should be added to indicate significant differences, and error bars should be included in related figures.

 

"Direct adventitious bulb induction not only shortens the production period but also prevents hyperhydricity, eliminates the need for hardening, and increases survival rate." There is no citation to support this claim. The study does not present data on hyperhydricity, making this statement speculative.

 

"This method can be successfully used to propagate T. tarda and other wild tulips." This statement is too broad: the study did not test other tulip species to justify this generalization. Additionally, it does not discuss study limitations, such as the lack of comparison with conventional propagation methods.

 

Of the 51 cited articles, 9 are from the same research group, which may indicate bias in the literature review.

 

Many references are over 10 years old and do not reflect recent advances in LED technology and new culture media formulations.

 

Miscontextualized Citation: "The mechanisms of in vitro storage organ formation in ornamental geophytes have been extensively reviewed [21]." Reference [21] (Podwyszyńska, 2012) does not comprehensively review these mechanisms; rather, it describes a specific study on ornamental geophytes.

Author Response

Dear reviewer,

I would like to thank you for sending me all the comments raised by you and the referees, which helped me to improve the manuscript.

 

I would like to explain as follows:

 

Comment 1: the title have been edited;

Comment 2: the abstract have been edited;

Comment 3: the statement about commercial production have been edited;

Comment 4: the number of explants per treatment was explained; according to LED light: I agree that there is no justification for using fluorescent light (instead of LED), but the experiment has already been performed under the fluorescent light, that is why I tried to find also publications using the fluorescent source of light;

Comment 5: the information about humidity and ventilation control is included;

Comment 6: the information about antioxidants is included;

Comment 7: the results have been discussed two lines below with previous studies with Tulipa tarda;

Comment 8: All data were checked for normality by the Shapiro-Wilk test;

Comment 9: The letters have been already included in the tables;

Comment 10: The word 'humidity' was removed from the manuscript;

Comment 11: The statement concerning using the method to other wild tulips was edited and focused only on T. tarda;

Comment 12: I cited the publications from the same research group, because I needed to compare the obtained results with the previous ones concerning T. tarda or other ornamental geophytes. However, I mostly cited publications by scientists from other centers;

Comment 13: I agree that I have cited references over 10 years, because such publications are still very important and sometimes the only available concerning ornamental geophytes, but I also cited publications from 2024 year - also concerning LED technology (eg. Pałka et al);

Comment 14: I did not discussed the mechanisms of in vitro bulb formation, but the individual factors influencing the formation of adventitious bulbs, that is why I have cited several times Podwyszyńska 2012.

Reviewer 3 Report

Comments and Suggestions for Authors

Title

You should edit the title to better match the research objective defined in lines 60-62, as well as the results obtained. (Lines 60-62. The research focused on determining the best light conditions, depending on the applied sucrose concentration and the presence of BAP, for the formation of T. tarda adventitious bulbs in vitro.)

The abstract is well written.

The introduction corresponds to the purpose of the study and the results obtained.

Material and methods are clearly described.

Results and discussion are very well written.

Results are very well presented in the three tables and well illustrated in the figures. The results proved that not only the light spectrum, but also the culture medium composition had significant effect on both induction of adventitious bulbs and number of bulbs per explant (P < 0.001), and that there was also significant interaction between the factors tested: light quality and culture medium composition (sucrose concentration and presence of BAP). The medium composition was not significant only concerning the induction and number of root and leaves; however, the interaction of the factors light and medium composition was proved (Table 3). The titles of the three tables are correct. The mutual influence of the factors light, BAP and sucrose concentration, are well described in the text as well, for ex. : line 116: “…the best treatment was white light combined with 6% sucrose”; line 155: “under white and blue light, the presence of BAP in the medium was necessary for root development”, etc.

So, the title of the manuscript should be edit to correspond to the results. Some possible titles could be for ex. “Effect of light quality and medium composition on in vitro adventitious bulb organogenesis of Tulipa tarda”, or “Interaction of light conditions and medium composition on in vitro adventitious bulb organogenesis of Tulipa tarda”.

The Conclusion corresponds to the results.

The combination of white light and 6% of sucrose resulted in 12.1 ± 1.3 bulbs per explant, as shown in Table 2, so you should replace 12 with 12.1 ± 1.3 or write “about 12” in the abstract (line 14) and in the conclusion (line 209). Same for the other average values.

Technical errors:

Line 74 – double comma

Line 116 – may be “it was observed” instead of “in was occurred”?

Line 120 – delete “of” or add “an average” before “of 7.6 buds per explant”

Line 183 – chose one of the following: “An adventitious bulb” or “Adventitious bulbs”

The English is very good.

Author Response

Dear reviewer,

I would like to thank you for sending me all the comments raised by you and the referees, which helped me to improve the manuscript.

 

I would like to explain as follows:

 

Comment 1: the title was edited;

Comment 2: the data contained in the Abstract and Conclusions were been clarified;

Comment 3: the technical errors were corrected.

Reviewer 4 Report

Comments and Suggestions for Authors

Comments concern:

1. Comparison of the obtained results primarily with the author's earlier publications [13,14]
2. How did the author determine true to type plants after 12 weeks of culture? Please remember that the culture was started from seed.
3. Unification of the font type in the References chapter

Author Response

Dear reviewer,

I would like to thank you for sending me all the comments raised by you and the referees, which helped me to improve the manuscript.

 

I would like to explain as follows:

 

Comment 1: the obtained results have been compared to the results from publication concerning T. tarda (previous results) in lines: 92, 111-112, 120-121, 133-136, 141, 146 – in scope: maximum adventitious bulbs per explant, influence of BAP, sucrose and blue light on number of the bulbs, influence of kind of explants on bulbs formation efficiency;

Comment 2: the true to type information was removed, due to initiation of culture from seeds,

Comment 3: the font type in References was unified