Echeveria Leaf Morpho-Anatomical Analysis and Its Implications for Environmental Stress Conditions

Reviewers

Reviewer Comments

Response

1s Reviewer

Dear Editor,

I reviewed the manuscript entitled Echeveria Leaf Morpho-Anatomical Analysis and its Implications for Environmental Stress Conditions by Tran et al.

Authors aim to elucidate leaf traits in Echeveria plants, especially those important for coping with climate change.

We are grateful for your comments and suggestions to improve and polish our study for publication. We have responded and made revisions based on your recommendations and we truly appreciate your time and effort spent on our paper.

I believe that MS can be of interest for the readers of Horticulturae working with succulents, even if English language needs a revision.

Furthermore, when describing and comparing species in anatomical and histological details, I strongly recommend including the plant material in resin and section it with an ultramicrotome so that the histological and anatomical traits are seen more clearly, and details are nice to appreciate.

Several changes are made to ensure the description of the method is accurate described. Also, the paper has undergone language editing outsourced by the authors (Editage.com) which we would gladly provide a certificate if needed.

In the recommendation of the use of resin in the sectioning, the authors chose to use Toluidine blue O (TBO) as the metachromatic staining, enabling distinct cells in a variety of colors, for ease of observation. The use of free-hand sectioning instead of ultramicrotome was also explained in L61-L73.

Introduction

The language can be improved but the content is coherent with the scope of the research.

Changes are made in L40-43, L39-42, L46-52, L54-57, L61-73, L 75-78, and L 93-94

Line 98

What do authors mean with “width thickness”? Maybe thickness of the leaf?

This was revised accordingly in L119.

Line 70 (from minor comment in pdf file)

Changes are indicated in L 93-94

Line 46 (from minor comment in pdf file)

This was revised accordingly in L 54 - 57

MM

I couldn’t find how authors made measurements of the leaf traits (e.g. epidermal and hypodermis layers thickness), this must be clarified.

Several changes are made to ensure the description of the treatment methods used. Revisions are indicated in L139 – L141.

3.2.1

I believe that all these data just reported between brackets need a graph to be visualized, and a statistical analysis done.

Changes are made in Fig. 2, and statistical analysis was done in Table 7, which included fundamental results from Table 3.

In Table 3, the results indicated the size of epidermis and hypodermis varies and having no visible trend between epidermis and hypodermis. Generally, the results showed that the enlarged cell size in hypodermis in most cultivars implied water storage function in these cells.

Fig 2k and l

Should be improved

Despite having leaf anatomical analysis has been studied widely in various species, there is limited research on the Echeveria genus particularly due to the lack of suitable methods. In this study, we are trying to employ the practical method using free-hand sectioning, which provides a relatively good visualization in transverse sections but is still restricted to some cultivars, which needs further analysis.

Line 253

I suggest to add in which of the fig.5 the 2d pattern is visible, and so also for the 3D.

Changes are indicated in Fig. 5

Discussion

Lines 339-341 are not clear.

Changes are indicated in L 400- 403

Reviewers

Reviewer Comments

Response

2^nd Reviewer

The manuscript is well-described with a clear aim and a significant conclusion drawn from the results. However, I have highlighted a few minor changes that need to be included before publishing your work. Kindly do the following correction 

We are grateful for your comments and suggestions to improve and polish our study for publication. We have responded and made revisions based on your recommendations and we truly appreciate your time and effort spent on our paper.

Title

In case you have studied more than one species in this study, mention Echeveria sp. or Morpho-Anatomical analysis of Genus Echeveria DC.

The sample plants used in this study are cultivars.

Page 2, Line 81:

Do these cultivars belong to any species? mention it,

This was revised accordingly in L 105

Page 2, Line 82:

Use author citation of the plants at their first mention Echeveria colorata E.Walther

This was revised accordingly in L 106

Page 3, Line 91:

What is the species of the studied plants?

Change is indicated in L 112

Page 3, Line 104:

What percentage is used?

Change is indicated in L 125

Page 3, Line 114:

Toluidine Blue O, and remove the following word in the sentence.

Change is indicated in L 136

Page 3, Line116:

Mention the camera system used to capture the image and the software used to analysis

Changes are indicated in L 135 – 140

Page 3, Line 118:

Leaf-Morpho-Anatomical observation or Leaf microscopy

Change is indicated in L 143

Page 4, Line 146:

Why is it in Bold?

This was changed accordingly.

Page 4, Line 150:

What about the species studied? Does it have trichomes?

Several changes are made and indicated in Table 1, L 182

Page 4, Line 162:

Use en dash (–): To indicate a range of numbers

Change is indicated in L 191

Page 5, In Section 3.2.1.

Epidermis and hypodermis. Are the values of epidermis and hypodermis in mm or µm? These characters are microscopic, so I'm not sure if they'll be in mm.

Changes are indicated in Table 3 in L 207 – 218

Page 5, Line 191

Please clarify the meaning of LM during the first introduction.

Several changes are made to ensure the description in L 61 -73

Page 5, Line 194

Repetition of Words

These terms are used to describe the periclinal cell walls and anticlinal cell walls. Changes are indicated in L 227 – 229

Page 5, Line 188

What are these cells? Which are enlarged in hypodermal layers?

Changes are indicated in L 221 -222

Page 5, Line 196

Specify which figure of Figure 3 has these characters.

Changes are indicated in L 227 -229

Page 6, Figure 2

Label which is epidermal cell and hypodermal cells

Changes are made in Fig 2, L 233

Page 7, Figure 3

periclinal and anticlinal wall of what?

Changes are made in L 241

In Figures 2 and 3, the magnification levels are different (100x and 200x) but the scale bar is the same. Please verify this.

Change is indicated in L 237

Page 8, Table 3:

Unit is doubtful. 

Changes are made in Table 3

Page 11, Line 275: 

It appears that observing a particular character in the figures is quite challenging. Typically, TBO stains purple or pink for the phloem and greenish or green-blue for the xylem.

However, in the figure, it is not possible to differentiate it. It is suggested that the author includes a close-up of the vascular bundles of each species, which would provide greater clarity.

Changes are indicated in Fig. 7

Abstract

Line 1:

Give the Family it belongs

Change is indicated in L12

Line 20:

what is the significant feature? Highlight your findings instead of saying the parameters

Change is indicated in L20- 22

Line 22:  

What is 3D vascular arrangements?

Change is indicated in L 23

Page 1, Line 41: 

Are there any studies on modifications to anatomy or external morphology such as size, shape, and thickness? Please mention if any exist.

Change is indicated in L 46

Page 1, Line 43:

What are enlarged vacuoles?

Several changes are made to ensure the description in L 46- 50

Page 2, Line 51: 

Add author citation.

Several changes are made to ensure the description in L 75 – 78

General comments

The usage of 2D and 3D to describe the vascular bundle is unsatisfactory. Because 2D and 3D are not descriptive here. 2D and 3D are dimension. The observations made using microscope images are 2D, not in 3D, hence they cannot provide an accurate representation of the vascular bundle in 3D. 

The best way of describing the vascular bundles is to determine what type of vascular bundle is present in each species studied. collateral or bi-collateral etc. 

We have responded and made revisions based on your recommendations, changes are made by adding Fig. 7.

Reviewers

Reviewer Comments

Response

3^rd Reviewer

1. The strong stress resistance of succulent plants is well-known, and many literature and plant physiology textbooks have detailed research or explanations. Their stress resistance mechanisms, such as drought resistance, are also manifested in multiple aspects, and morphological characteristics are only one of them, including stomatal opening and closing, photosynthetic pathways, root characteristics, and other aspects. Therefore, from this perspective, the innovation of this article is not strong.

We appreciate your insightful comments and would like to supplement our response by providing additional context regarding the unique challenges and contributions of our research on succulent plants, particularly within the context of the Echeveria genus belonging to the Crassulaceae family.

Succulent plants, such as those in the Echeveria genus, are renowned for their adaptation to arid environments through a combination of morphological and anatomical features. These adaptations have evolved to efficiently utilize water, making them well-suited for growth in challenging conditions. Moreover, the increasing demand for succulents in horticultural markets, especially the growing variety of Echeveria cultivars, highlights their popularity as ornamental plants. Their xeromorphic features and aesthetic appeal make them ideal for urban areas where greenhouse space is limited, and citizens seek low-maintenance yet visually appealing greenery.

However, the surge in cultivar diversity has posed challenges in identification and classification due to homoplastic morphological features. Despite similar external morphology, individual cultivars may exhibit distinct responses to external conditions, adding complexity to their management.

Anatomical studies of these plants face limitations due to thick cuticles, the presence of tannins, and high mucilage content. These factors hinder comprehensive understanding, making it imperative to develop accessible and less time-consuming methods for studying succulent anatomy, especially in controlled conditions.

Our research addresses these challenges by exploring the intricate relationship between morphological features and stress resistance in succulent plants, with a specific focus on the Echeveria genus. By providing a methodological framework that is both accessible and time-efficient, our study aims to enhance the understanding of physiological aspects and relationship dynamics among different cultivars. These results are not only beneficial for the scientific community but also provide crucial information for breeders and plant growers. This knowledge empowers them to optimize conditions for individual plants in greenhouse settings and offer valuable advice to customers, contributing to the sustainable cultivation and appreciation of succulent varieties.

We hope this additional information provides a more comprehensive perspective on the significance and innovation of our research. Your feedback has been instrumental in refining our manuscript, and we are grateful for your thoughtful evaluation.

2. Can the results of the difference significance analysis or coefficient of variation be added to Tables 2, 3, 5, and 6 to represent the inter variety differences of a certain indicator.

Based on our findings, the majority of the examined cultivars manifest homoplasy, displaying xeromorphic features, a phenomenon deemed of nominal significance within this study. Nevertheless, specific parameters, notably stomatal density, leaf thickness, and moisture content, have emerged as discernible indicators that bear the potential to augment comprehension. These identified indicators offer a pathway to refine our understanding and, notably, optimize conducive conditions tailored to the unique requirements of individual plants within this taxonomic cohort.

3. The author only used the relevant indicators of free hand sectioning slices to evaluate stress resistance, and the workload was slightly less. Has the structural characteristics of leaf transverse or longitudinal sections and their differences among varieties been observed.

The outcomes derived from transverse sections and anatomical analyses furnish foundational insights crucial for the refinement of external conditions during the cultivation of these cultivars within controlled environments. The escalating demand for these plants notwithstanding, the paucity of information pertaining to the care of specific cultivars is underscored by our findings, elucidating the divergent responses exhibited by individual plants within varying environmental contexts, as elucidated in L 461-463 and L 481-484.

4. The author's contribution can be simply written. With only three people, there is no need for the author's division of labor to be so complex.

The authors’ contribution was made based on the journal’s format.