Plant Tissue Culture In Vitro: A Long Journey with Lingering Challenges

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Pasternak and Stenmacher describes strategies to improve efficiency in in vitro plant tissue culture. By examining the role of auxin and nutrient balance in micropropagation, the authors emphasize the importance of establishing species-specific culture conditions. I agree with their main argument and believe that it reflects an important viewpoint in recent plant biology. However, the claims presented in the manuscript are not sufficiently supported by the figure, text, or appropriate citations. In its current form, the manuscript lacks the clarity and credibility. Specific issues are outlined below.

Major points

1. Abstract and main text are not well aligned. The abstract is attractive, particularly in how it criticizes the modern practice deeply dependent on traditional protocols and emphasizes the necessity of species-specific optimization. I fully support this perspective. However, the main text does not present data or citations that substantiate these claims (see also comment 2 below).

In fact, the manuscript primarily discusses the role of auxin metabolism and its regulation by nutrient status during regeneration. The authors should revise either the abstract or the main text so that the two are clearly aligned in scope and content.

Exapmle1: L133-134: An optimized medium with balanced nutrient composition is essential for improving micropropagation efficiency and plant quality [25]”

Reference 25 is a paper about YUC gene function in Arabidopsis development, and it does not address culture optimization or micropropagation. A more relevant citation is needed.

Example 2: L103-L106 “Such formulation, known as MS, … as soft callus formation and excessive water uptake [30].”

This argument is currently supported by a single review article [30]. It would be more informative if the authors cited original research articles providing examples of species or systems in which MS medium does not work well.

2. There are some fact statements that lack citations. The reviewer requests that the authors add appropriate references to the sentences shown below to ensure the trustability and traceability of the manuscripts

L80 In this context,…: No citation

L92 In this direction,…: No citation

L93 Root induction requires…: No citation

L95 Only after…: Murashige and Skoog (1962) is cited, but does the original MS paper really discuss the “conditions,” as the authors implied, under which exogenous auxin supports de novo root formation?

L113-L117: No citation

L119 - L125: No citation

3. The current figure does not function effectively as a scientific illustration. It is difficult to interpret and provides little information. In Panel A, the chemical structure is not labeled, and its relationship to the text and symbols is unclear. A text is written in green font on a green background, making it unreadable. The flower icon at the bottom is also unexplained. In Panel B, only a list of keywords is shown. If the authors intend to represent a timeline or a process, then using icons such as arrows would help clarify the intended sequence or relationship.

4. Several sentences are difficult to understand due to missing or duplicated subjects or verbs. Language editing is recommended, particularly for the following sentences:

L45-L47 Recent in situ data…

L55-L57 Interesting, that only…

L146-147 The traditional theory emphasizes…

Minor points & typos

L82, L113: Remove unnecessary parentheses: "in planta)", "well)"

L96: Please define the abbreviation “PTC”

L97–101: I was a little confused because the term “generative growth” typically refers to reproductive development (flowers and seeds), which does not usually occur in tissue culture. Please clarify the definition of this term or cite literature that uses it in the context of tissue micropropagation.

L118: “As” → possibly a typo for “A”?

L132–133: Please format the citation properly

Comments on the Quality of English Language

See Major point 4.

Author Response

Thank you very much for reviewing our opinion paper and very constructive suggestions.

We have revised text accordingly to your suggestions.

Sincerely.  Dr. Taras Pasternak; Dr. Douglas Steinmacher

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled "Plant tissue culture in vitro: a long journey with lingering challenges" provides a valuable perspective by highlighting the significant roles of endogenous auxin synthesis and epigenomic status in plant morphogenesis. The authors rightly propose that the traditional theory, which focuses primarily on the exogenous auxin/cytokinin balance, requires re-evaluation.

However, the manuscript's argument could be significantly strengthened by adopting a more integrated view. While the focus on endogenous auxin is a critical advancement, the discussion largely overlooks the equally vital involvement of other phytohormones, such as cytokinins and gibberellins, in controlling cell division, differentiation, and morphogenesis.

Therefore, I suggest that the call to re-evaluate the traditional theory should be expanded. To be truly comprehensive, a new model must not only incorporate endogenous auxin synthesis and epigenetics but also map the complex crosstalk, synergy, and antagonism between these factors and other critical hormone signaling pathways. Including a discussion on this broader hormonal network would provide more compelling evidence and substantially elevate the impact of the manuscript.

In addition to the major points above, several minor corrections are needed to improve the manuscript's clarity and professionalism:

1. Line 21: Please correct the typo "moder biotechnologies" to "modern biotechnologies."
2. Line 54: A period (.) is missing at the end of the sentence, after the citation [12-16].
3. Subheading 2: The parenthetical phrase (....and the limitations of MS) appears to be incomplete. Please check for missing text before "and" or clarify the intended meaning.
4. Line 137: The abbreviation "TK" is used without being defined. Please provide the full term upon its first use.
5. Line 140: The scientific name Arabidopsis should be italicized, following standard convention.
6. Figure Citations: The format for citing figures in the text is inconsistent (e.g., "Figure 1" vs. "Fig. 1"). Please choose one format and apply it uniformly throughout the manuscript.
7. In-text Reference Citations: The manuscript mixes different citation styles (e.g., numerical [1] and author-text styles). Please adhere to a single, consistent citation format as required by the journal.
8. Reference List Formatting: The style for journal names in the reference list is inconsistent, using both full names and abbreviations. Please format all entries according to the journal's specific guidelines.

Author Response

Thank you very much for reviewing our opinion paper and very constructive suggestions.

We have revised text accordingly to your suggestions.

Sincerely.  Dr. Taras Pasternak; Dr. Douglas Steinmacher

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for revising the text, I have no more comments to add.

Author Response

Thank you very much for your time and efforts. My best regards!

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed almost all comments, but the reference list still requires formatting. Some references remain incorrect. Please correct references 14, 16, 28, 31, 37, and 40.

Author Response

Thank you very much for your efforts and time for evaluation of our manuscript. We have adjusted citations. My best regards!