Micropropagation from Inflorescence Nodal Segments of Phalaenopsis and Acclimatization of Plantlets Using Different Substrates
, Willian Naves Duarte 1, Daniela Mangueira Gomes 2 and Jean Carlos Cardoso 3,*
Round 1
Reviewer 1 Report
Dear Authors,
The introduction section is very well written, covering all important aspects of Phalaenopsis as an ornamental cut or potted plant, introducing problems related to the propagation and leading to the research aim.
M&M section is very well organized, with sufficient explanations. Some minor comments include:
Line 88 Please provide the full name for New Dogashima Medium, since it is first mentioned here (excluding the mentioning in the abstract).
Line 113 ‘An additional experiment was also carried out with the cultivar Phalaenopsis‘PH501’ 113 in order to increase the yield of shoots obtained by subculture cycle.’ Please explain why you selected this cultivar, over 'PH908', as it was explained in line 256.
Line 139 ‘The acclimatization was realized in plastic trays..’ please find a more appropriate term for realized, like performed or similar.
Line 151 Please add a period at the end of the sentence.
Results and discussion.
Line 162. I wouldn’t recommend the start of this important section with exclusively negative results. Please rephrase and reorganize lines 162 – 173.
Line 232 – 233 please improve the English language here to make sentences more understandable.
Line 283, please write the full name for Phal. amabilis.
Line 332 and 339 again please avoid term ‘realized’, and use the more suitable word as achieved, performed and similar.
Line 358 ‘Also, we were obtained an important correlation between the plantlets development and clorophyll indices’. Delete the word ‘were’.
Line 367 correct this part, there is unnecessary bracket ‘(1:1). ),’
The conclusion section is very well written.
Author Response
Dear Reviewer 1, we would like to thanks you for changes required and contributions to our manuscript. We hope we attend your queries in this new reviewed version. Best regards, Jean Cardoso
Reviewer 1
Dear Authors,
The introduction section is very well written, covering all important aspects of Phalaenopsis as an ornamental cut or potted plant, introducing problems related to the propagation and leading to the research aim.
M&M section is very well organized, with sufficient explanations. Some minor comments include:
Line 88 Please provide the full name for New Dogashima Medium, since it is first mentioned here (excluding the mentioning in the abstract).
Response: the name was provided
Line 113 ‘An additional experiment was also carried out with the cultivar Phalaenopsis‘PH501’ 113 in order to increase the yield of shoots obtained by subculture cycle.’ Please explain why you selected this cultivar, over 'PH908', as it was explained in line 256.
Respose: This information was provided
Line 139 ‘The acclimatization was realized in plastic trays..’ please find a more appropriate term for realized, like performed or similar.
Response: we described with more detais the plastic trays, as follow: 50-cells (40 mm x 40 mm x 90 mm depth) plastic trays
Line 151 Please add a period at the end of the sentence.
Response: This information was improved in the sentence
Results and discussion.
Line 162. I wouldn’t recommend the start of this important section with exclusively negative results. Please rephrase and reorganize lines 162 – 173.
Response: We agree and we reordered the sentences to provide positive results instead of negative explanations
Line 232 – 233 please improve the English language here to make sentences more understandable.
Response: The sentence was rewritten
Line 283, please write the full name for Phal. amabilis.
Response: ok
Line 332 and 339 again please avoid term ‘realized’, and use the more suitable word as achieved, performed and similar.
Response: This term was changed for other more appropriate in the sentences.
Line 358 ‘Also, we were obtained an important correlation between the plantlets development and clorophyll indices’. Delete the word ‘were’.
Response: We re-written this sentence as follow: Also, important correlation between the plantlets development and clorophyll indices was reported.
Line 367 correct this part, there is unnecessary bracket ‘(1:1). ),’
Response: corrected
The conclusion section is very well written.
Thanks for all considerations and improvements in our manuscript.
Reviewer 2 Report
Horticulturae-1633170
The title should change to "Clonal micropropagation of Phalaenopsis from inflorescence nodal segments and acclimatization of plantlets under different substrates".
- The English has to be improved by a native English speaker.
Introduction (please cite MDPI journal as much as possible):
- Line 48 or line 61 to 66: The authors note the usage of inflorescence stalks without mention of the problems flowering causes in regeneration (https://www.mdpi.com/2079-7737/10/9/937) and how to overcome them (https://www.nature.com/articles/s41598-021-93180-1; https://www.mdpi.com/2223-7747/10/1/58)
- What is unique about this manuscript? It is unclear why the work was done from the introduction.
Results and Discussion:
- Notification on Figure 1 is missing, no E, for example.
- Line 206-207, an explanation why after the first passage on medium, only vegetative shoots are formed and not inflorescence?
- There are no meaningful differences in table 2. It could be just stated in the text.
- Line 324: seddling?
The manuscript is very technical with no scientific value. The discussion should deal with possible explanations to problems or conflicts between the authors and other published data; why BA behaves differently in their system, why GA has an effect sometimes, why inflorescence and not side buds as starting material?
Author Response
Reviewer 2
The title should change to "Clonal micropropagation of Phalaenopsis from inflorescence nodal segments and acclimatization of plantlets under different substrates".
Response: The title was modified according the two suggestions by reviewer 2 and 3.
- The English has to be improved by a native English speaker.
Response: The English language have revised and edited by a native English Speaker
Introduction (please cite MDPI journal as much as possible):
- Line 48 or line 61 to 66: The authors note the usage of inflorescence stalks without mention of the problems flowering causes in regeneration (https://www.mdpi.com/2079-7737/10/9/937) and how to overcome them (https://www.nature.com/articles/s41598-021-93180-1; https://www.mdpi.com/2223-7747/10/1/58)
Response: We would like thanks for your suggestion and this is a ‘giant’ discussion in plant biology, and we add in this section the following sentence:
Some plant genotypes presented a large recalcitrance for regeneration when flower-ing occurred (Reuveni, 2021), with recent studies appointing a possible interface between the florigen mRNA and the inhibition of tissue/organ regeneration (Kutsher et al. 2021). However, in plants, flowering development were divided into two main stages: the flow-ering induction and the effective development of inflorescences and flowers. The develop-ing or developed floral organs and tissues have been used aiming in vitro regeneration and present until now interesting applications in different important crops, such as the induction and regeneration of somatic embryogenesis pathway (Almeida et al. 2022; Car-doso et al. 2017). In addition, floral organs in some species, is the unique viable explant using as alternative to apical shoot meristems, such as in Phalaenopsis, a monopodial orchid which the assessment of shoot tips result into the death of the mother plant. Thus, in Phalaenopsis the previous-developed buds presented in the nodes of flowering stalks, make this tissue a realistic method for shoot induction, by the activation of previously formed-buds contained in each node used as explant (Cardoso and Zanello, 2018).
- What is unique about this manuscript? It is unclear why the work was done from the introduction.
Response: The most of in vitro protocols used model-species of Phalaenopsis to test the regeneration and shoot proliferation. However, we observed that these protocols are limited to these genotypes, normally obtained from in vitro seedlings, which have no practical horticultural interest aiming clonal propagation of Phalaenopsis. Our paper showed the results of commercial hybrids really used to produce flowers and shown the main factors affecting these in vitro cultivation systems that are obtained from somatic tissues (inflorescences) from greenhouse cultivated mother plants. Also, we reported the main limitations and factors lead to a successful protocol for clonal propagation of the most economically important orchid in the world floriculture.
To provide this information in the manuscript, we added the follow sentence before the objectives:
The main questions to be responded with this paper are: Are the commercial hybrids of Phalaenopsis responded at the same level of model-species, such as Phal. amabilis conventionally used for in vitro regeneration? Are there a specific importance of different phyto-regulators classes, such as GA3 and 6-BA on bud sprouting and shoot proliferation of Phalaenopsis hybrids? 6-BA used in multiplication phase have residual influences on plantlet development in rooting phase? What changes occurred in acclimatization of Phalaenopsis clonal plantlets when acclimatized using the most common substrates in world floriculture?
Results and Discussion:
- Notification on Figure 1 is missing, no E, for example.
Response: Ok, corrected
- Line 206-207, an explanation why after the first passage on medium, only vegetative shoots are formed and not inflorescence?
Response: This discussion was added to the manuscript, with references additions.
- There are no meaningful differences in table 2. It could be just stated in the text.
Response: The table 2 present detailed analysis with significative differences among treatments and was important to describe the main factors associated with each phytorregulator on multiplication phase. There are two variables added to this table, and the differences between letters are presented in each column of the table, as described in table footnote.
- Line 324: seddling?
Response: Corrected, thanks!
The manuscript is very technical with no scientific value. The discussion should deal with possible explanations to problems or conflicts between the authors and other published data; why BA behaves differently in their system, why GA has an effect sometimes, why inflorescence and not side buds as starting material?
Response: We agree that this article have a technical content which could be used direct in commercial labs. To provide and increase scientific value, we increased all information you requested, but also avoiding extensive discussions with can not proved by our results. I hope we attend your main queries for increase the scientific value of the paper, by increasing informations in R&D section. As example, we added information about inflorescence and vegetative development of Phalaenopsis based in BA experiments, and also the physiological effects of BA on in vitro development and we added a sentence about the correlation between GA3 addition and browning of the tissues, normally affecting Phalaenopsis micropropagation.
About not use side buds, Phalaenopsis is a monopodial plant with undeveloped pseudobulb. The accession of side and apical bud lead to the death of mother plants. Thus, inflorescence segments are a non-destructive alternative that also leads to efficient shoot proliferation of Phalaenopsis. This information was efforted in the manuscript.
Also, please, if you have any other specific query we are try to improve with a pleasure.
Best regards and thanks for your detailed review.
Reviewer 3 Report
The manuscript reports an in vitro propagation method of hybrids of a commercially important species, therefore it may have an important applicative interest, it does not present any significant novelties from the point of view of the applied in vitro culture methods which are very traditional, it is therefore necessary that the authors are able to highlight well the originality that is not perceived in this version.
It can only be published after a rewrite based on the following general considerations:
- change the title, focusing it better
-the introduction rather than repeating well-known things about orchids, should highlight the specificity of the proposed protocol with respect to the traditional propagation through protocorms and / or if it can be connected to it.
-The methods must be reported with more precision eliminating the errors
-The results and discussion must be reported with greater clarity and conciseness, aiming at the essential results.
Writing in English must be drastically corrected.
I report a list of more detailed suggestions which are also reported as notes in the text
Change the title: Inflorescence nodal segments and derived-shoots for clonal micropropagation of Phalaenopsis and acclimatization of plantlets under different substrates.
Micropropagation is always a clonal multiplication technique, needless to say; acclimatization is performed using or inside different substrate not under
Material and methods: in general, better clarify the text, verify chemical nomenclature and citation
Line 120: if this method to prepare the explants is efficient and new, why does not report as a result? This could improve the originality of the paper.
Line 136: better specify medium composition: have you used MS macro elements at half strength, but micro elements? You have only added inositol or even the other vitamins provided in the MS formulation
Line 150: Attention!!!!Chlorofilometer is an instrument to evaluate chlorophyll content ( expressed as an index) but not the chlorophyll fluorescence...
Results and discussion:
In fig 1 you attribute the contamination to the presence of yeasts, in the text it is not mentioned, have you made some microbiological characterizations? If you have not done them, it is better not to declare the origin of the pollution
Line 218 220 the conclusion is wrong, only the mean with BA1 and 0.15 GA3 is greater than the control (means with letters ab and b must be considered equal)
Line 226 the discussion must be reformulated, it is obvious that in vivo treatments to induce flowering cannot be compared with the use of GA3 in an in vitro culture medium .... moreover if it had stimulated real inflorescences it would not have been a positive result because purpose is to produce non-inflorescence shoots .... I would just say no differences attributable to the different concentration of GA3 were observed. The experimental plan adopted does not even allow to evaluate a possible positive effect of GA3 on the stimulation of dormant buds because it is present in all three media used.
3.1 paragraph: the discussion of these first results must be rewritten, discussing only the stastically different results, the authors must clarify, on the basis of the literature if in this initial phase of the culture it is important to have viable explants even with low development of sprouts otherwise the results obtained are inconsistent and they can only be considered foreplay
Fig.2: change the axis label
Line 310: better explain the results. an average values less than 1 root for plant means that some plants are without roots? and if so, how is possible that these plants without roots and also without leaves showed 100% acclimatization?
Line 316 TAB2: you report an index of chlorophyll content not parameters relative to chlorophyll fluorescence!!!!see this rew https://doi.org/10.1093/jexbot/51.345.659
Measurements of chlorophyll a fluorescence can be conducted using a chl fluorometer
Line338: reduce the discussion about the effect of different substrate, many concepts are repetitive, better limiting to cite the principal factor involved in the response of plants to the various culture condition and also the genotype differences
Comments for author File: 
Comments.pdf
Author Response
Reviewer 3
Comments and Suggestions for Authors
The manuscript reports an in vitro propagation method of hybrids of a commercially important species, therefore it may have an important applicative interest, it does not present any significant novelties from the point of view of the applied in vitro culture methods which are very traditional, it is therefore necessary that the authors are able to highlight well the originality that is not perceived in this version.
Response: We like to thanks you for commentaries and important improvements to our paper. We try to change some parts of introduction and added information about highlights and main questions to be responded with the experiments conducted. In addition, we added new separate results and discussion sections to provide better meaning about our main findings. We hope that manuscript meet your requests. Also, we provided the detailed information and review using the PDF you attached in the system. Thanks and best regards,
It can only be published after a rewrite based on the following general considerations:
- change the title, focusing it better:
Response: the title was modified according suggestions of the three reviewers
-the introduction rather than repeating well-known things about orchids, should highlight the specificity of the proposed protocol with respect to the traditional propagation through protocorms and / or if it can be connected to it.
Response: We added this information in two different sentences: one discussing the use of Protocorm-Like Bodies in this genus and other with the specificity the limitations and advantages of the technique of direct shoot proliferation, as follow:
Protocorm-like Bodies (PLBs) derived from different types of somatic tissues is one clonal method used to produce large quantities of plantlets from few mother plants (Tokuhara and Mii, 1993; Zanello and Cardoso, 2018). However, the regeneration of PLBs from different tissues and in different genotypes have showed as main limitation the occurrence of different frequencies (0–51.7%) of somaclonal variations (SVs), with reported SVs in flower development, e.g. absence of flower parts of Phalaenopsis ‘PH908’ (Cardoso et al. 2020).
Some plant genotypes presented a large recalcitrance for regeneration when flowering occurred (Reuveni, 2021), with recent studies appointing a possible interface between the florigen mRNA and the inhibition of tissue/organ regeneration (Kutsher et al. 2021). However, in plants, flowering development were divided into two main stages: the flowering induction and the effective development of inflorescences and flowers. The developing or developed floral organs and tissues have been used aiming in vitro regeneration and present until now interesting applications in different important crops, such as the induction and regeneration of somatic embryogenesis pathway (Almeida et al. 2022; Cardoso et al. 2017). In addition, floral organs in some species, is the unique viable explant using as alternative to apical shoot meristems, such as in Phalaenopsis, a monopodial orchid which the assessment of shoot tips result into the death of the mother plant (Chugh et al. 2009). Thus, in Phalaenopsis the previous-developed buds presented in the nodes of flowering stalks, make this tissue a realistic method for shoot induction, by the activation of previously formed-buds contained in each node used as explant (Tokuhara and Mii, 1993; Cardoso and Zanello, 2018).
-The methods must be reported with more precision eliminating the errors
Response: We try to add some missed informations along Materia and Methods. Please, if you identified any additional specific error please inform us.
-The results and discussion must be reported with greater clarity and conciseness, aiming at the essential results.
Writing in English must be drastically corrected.
I report a list of more detailed suggestions which are also reported as notes in the text
Response: We try to meet your queries and also by Reviewer 2, which required additional information to increase the scientific quality of the discussion section. In addition, we separated the both sections into two: Results section and Discussion Section to provie improvements and concentrate information and meaning. In addition, the English was revised by an expert in English language to improve the quality of the paper.
Change the title: Inflorescence nodal segments and derived-shoots for clonal micropropagation of Phalaenopsis and acclimatization of plantlets under different substrates.
Micropropagation is always a clonal multiplication technique, needless to say; acclimatization is performed using or inside different substrate not under
Response: The title was changed and include suggestions by you and reviewer 2.
Material and methods: in general, better clarify the text, verify chemical nomenclature and citation OK
Line 120: if this method to prepare the explants is efficient and new, why does not report as a result? This could improve the originality of the paper.
Response: Thanks for this insight and we agree that this information is major interesting in Results, as follow:
Using shoot proliferation, all the leaves were excised, keeping only the apical region containing the leaf primordia and the proximal region of developed leaves, instead of intact plants. This procedure has been performed and promoted a greater number of shoots from the axillary buds than intact plants (data not shown).
Line 136: better specify medium composition: have you used MS macro elements at half strength, but micro elements? You have only added inositol or even the other vitamins provided in the MS formulation
Response: We only reduced the macroelements, maintaining all of the other elements in its original concentration.
Line 150: Attention!!!!Chlorofilometer is an instrument to evaluate chlorophyll content ( expressed as an index) but not the chlorophyll fluorescence...OK
Results and discussion:
In fig 1 you attribute the contamination to the presence of yeasts, in the text it is not mentioned, have you made some microbiological characterizations? If you have not done them, it is better not to declare the origin of the pollution
Response: We corrected this part and add some information in discussion section.
Line 218 220 the conclusion is wrong, only the mean with BA1 and 0.15 GA3 is greater than the control (means with letters ab and b must be considered equal)
Response: We worked in this conclusion to improve the meaning.
Line 226 the discussion must be reformulated, it is obvious that in vivo treatments to induce flowering cannot be compared with the use of GA3 in an in vitro culture medium .... moreover if it had stimulated real inflorescences it would not have been a positive result because purpose is to produce non-inflorescence shoots .... I would just say no differences attributable to the different concentration of GA3 were observed. The experimental plan adopted does not even allow to evaluate a possible positive effect of GA3 on the stimulation of dormant buds because it is present in all three media used.
Response: We agree! This discussion about correlation of ex vitro plants and in vitro tissues was deleted from the text and was concentrated on the effect of genotype inflorescence development, with no effects of phytorregulators.
3.1 paragraph: the discussion of these first results must be rewritten, discussing only the stastically different results, the authors must clarify, on the basis of the literature if in this initial phase of the culture it is important to have viable explants even with low development of sprouts otherwise the results obtained are inconsistent and they can only be considered foreplay
Response: We revised these parts and rewritten the most part of results and discussion based on the statistical analysis of the tables and figures.
Fig.2: change the axis label
Response: The axis label was modified considering all the variables analyzed
Line 310: better explain the results. an average values less than 1 root for plant means that some plants are without roots? and if so, how is possible that these plants without roots and also without leaves showed 100% acclimatization?
Response: Please, see that these values used in this table is the gain in leaves and roots, and after 60-d greenhouse cultivation, compared to initially acclimatized plantlets. Thus, 0,5 represents the gain in mean of 0,5 leaf for each plantlet acclimatized.
Line 316 TAB2: you report an index of chlorophyll content not parameters relative to chlorophyll fluorescence!!!!see this rew https://doi.org/10.1093/jexbot/51.345.659
Response: The terms were replaced in the text and discussion including this review and the main concepts of Chlorophyll fluorescence measurements.
Measurements of chlorophyll a fluorescence can be conducted using a chl fluorometer
Line338: reduce the discussion about the effect of different substrate, many concepts are repetitive, better limiting to cite the principal factor involved in the response of plants to the various culture condition and also the genotype differences
Respose: We agree and this part was several reduced, maintaining only the main findings about Phalaenopsis acclimatization.
Thank you very much for your valuable support and careful revision of our manuscript. Jean Cardoso
Round 2
Reviewer 2 Report
Your corrections are adequate for publication
Reviewer 3 Report
The manuscript has been drastically corrected following my suggestions starting from the title and correcting the methods. The results were rewritten respecting the reported data and the discussion was better focused.
