In Vitro Micropropagation, Rooting and Acclimatization of Two Agastache Species (A. aurantiaca and A. mexicana)
, Paolo Mussano 1,2, Poonam Devi 1,3, Angelo Lanteri 1, Arianna Cassetti 1, Carlo Mascarello 1, Angela Bisio 3 and Barbara Ruffoni 1Round 1
Reviewer 1 Report
The article considers the methodological aspects of in vitro propagation and acclimatization of two varieties of Agastache (A. aurantiaca ‘Sunset Yellow’ and A. mexicana ‘Sangria’). The article contains new experimental data on micropropagation, rooting and acclimatization of these food plants in in vitro culture, which contribute to an increase in the amount of planting material. The article is well documented with photographs and experimental data that reasonably prove the main points made by the authors.
I submit that after correcting some errors and incomplete information (listed below), the article can be recommended for publication.
Notes:
1. Lines 77-83: provide links to the reported micropropagation of Agastache in vitro. Perhaps the authors should note the shortcomings of previous studies with these plants and the need to establish a new propagation protocol.
2. L. 101-102: specify that the microcuttings were with buds (nodal cuttings).
3. L. 117: what does [x] mean?
4. L. 117-118 - repeat of the BAP decoding.
5. L. 154, 186 - incorrect numbering of sections. It is necessary to add to the title of the section (on L. 154) ‘ex vitro’.
6. L. 173-176 and 183-184 - repetition of information.
7. L. 182 - decoding of TBO is required.
8. Tables 2 and 3 - it should be clarified that different letters are used for different treatments and for different Agastache species.
9. I would recommend (in section 5. Conclusion or at the end of section 4) giving an estimate of the number of new plants and flowers resulting from the use of the developed protocol of in vitro micropropagation.
Author Response
Answers - Reviewer 1
Notes:
- Lines 77-83: provide links to the reported micropropagation of Agastache in vitro. Perhaps the authors should note the shortcomings of previous studies with these plants and the need to establish a new propagation protocol.
Answer: This part has been added - Indeed, Carmona Castro et al. [12] focused their study on the production of secondary compounds from biomass in vitro and obtained poor results during the acclimatization phase: 33% of plants survived.
- L. 101-102: specify that the microcuttings were with buds (nodal cuttings).
Answer: This part has been added “…with 2-4 nodal buds and without leaves”
- L. 117: what does [x] mean?
Answer: citation has been added
- L. 117-118 - repeat of the BAP decoding.
Answer: the repetition has been eliminated
- L. 154, 186 - incorrect numbering of sections. It is necessary to add to the title of the section (on L. 154) ‘ex vitro’.
Answer: the number of sections has been corrected
- L. 173-176 and 183-184 - repetition of information.
Answer: this part has been modified by removing the repetition
- L. 182 - decoding of TBO is required.
Answen: Toluidine Blue O stain has been added
- Tables 2 and 3 - it should be clarified that different letters are used for different treatments and for different Agastache species.
Answer: in both tables the sentence has been added “No comparisons have been made between species.”
- I would recommend (in section 5. Conclusion or at the end of section 4) giving an estimate of the number of new plants and flowers resulting from the use of the developed protocol of in vitro micropropagation.
Answer: This part has been added at the end of section 3.3 - In fact, most of the clusters grown in the greenhouse produced apical inflorescences and the clusters with more shoots (from TIS culture) produced on average almost twice as many inflorescences than the clusters with fewer shoots (from solid medium) (data not shown).
In the article, the corrections are underlined in blue
Reviewer 2 Report
The article "In vitro micropropagation, rooting and acclimatization of two Agastache species (A. aurantiaca and A. mexicana)" is dedicated to development of an efficient clonal micropropagation protocols for two Agastache aromatic species (A. aurantiaca (A. Gray) Linton & Epling and A. mexicana (Kunth) Lint & Epling). Experimental data are characterized by novelty and relevance. Correct statistical processing of experimental data was carried out. The manuscript is well illustrated. I have a few comments and questions about this manuscript.
1) Add of the author-classifier to the genus and species names of A. aurantiaca (A. Gray) Linton & Epling and A. mexicana (Kunth) Lint & Epling.
2) L.97. 9 l of volume. It is not clear what the authors meant.
3) Figure 1. I think the figure 1 is meaningless.
4) Figures 2, 3 and 6 require significant improvement.
5) The bar in figure 7 is not visible.
6) Figure 8. What is the meaning and what differences are there between the photos on the Fig.8 A and A’, and B and B’?
7) Conclusion. How long does it take for a full cycle of clonal micropropagation from obtaining aseptic explants to plant acclimatization for soil conditions? You can specify the duration of each stage. What level of multiplier coefficient or rate has been obtained using this protocol?
Best regards,
reviewer.
Author Response
Reviewer 2
The article "In vitro micropropagation, rooting and acclimatization of two Agastache species (A. aurantiaca and A. mexicana)" is dedicated to development of an efficient clonal micropropagation protocols for two Agastache aromatic species (A. aurantiaca (A. Gray) Linton & Epling and A. mexicana (Kunth) Lint & Epling). Experimental data are characterized by novelty and relevance. Correct statistical processing of experimental data was carried out. The manuscript is well illustrated. I have a few comments and questions about this manuscript.
1) Add of the author-classifier to the genus and species names of A. aurantiaca (A. Gray) Linton & Epling and A. mexicana (Kunth) Lint & Epling.
Answer: author-classifier of Agastache genus was added in the introduction
2) L.97. 9 l of volume. It is not clear what the authors meant.
Answer: the capacity of the pot is 9 litres; the diameter of the vase is 30 cm
3) Figure 1. I think the figure 1 is meaningless.
Answer: figure 1 shows the reader the type of jar used, showing the size of the microcuttings and the distance of the microcuttings in the jars
4) Figures 2, 3 and 6 require significant improvement.
Answer: to improve observation of the images, they could be larger
5) The bar in figure 7 is not visible.
Answer: we can try changing the color of the bars from red to black
6) Figure 8. What is the meaning and what differences are there between the photos on the Fig.8 A and A’, and B and B’?
Answer: A and B: leaf section of A. aurantiaca; A’ and B’: leaf section of A. mexicana; A and A’: leaves from jar; B and B’: leaves from in vivo plants. The leaves from both jar had thinner epidermis cell walls and waxes, less extensive and organized palisade parenchyma, and looser spongy tissue than those from leaves harvested from in vivo plants.
7) Conclusion. How long does it take for a full cycle of clonal micropropagation from obtaining aseptic explants to plant acclimatization for soil conditions? You can specify the duration of each stage. What level of multiplier coefficient or rate has been obtained using this protocol?
Answer: Timings regarding micropropagation, rooting and acclimatization have been added to the text. The multiplication coefficient was not calculated
In the article, corrections are underlined in green
Reviewer 3 Report
The manuscript describes a study comparing various treatments aimed at inducing in vitro shoot development and subsequent rooting of two species within the Agastache genus. One important result was the identification of an optimal 6-benzyl amino purine (BAP) concentration to induce shoots and explants suitable for subsequent rooting. For rooting, the study investigated two treatments, one involving traditional culture in vessels with agar and another using a bioreactor where the explants were exposed intermittently to a liquid medium. Both methods yielded a high frequency of rooted explants, but the bioreactor technique produced larger plantlets with more roots and shoots. Plantlet survival was very high during the acclimation stage, independent of the rooting method. However, plantlets from the bioreactor maintained and increased their characteristics of being larger and with more shoots. I believe the protocols reported will benefit researchers or growers interested in the clonal multiplication of the studied species or other species with the same family. Furthermore, the bioreactor method employed is not so well known. The manuscript may contribute to expanding the use of this method and testing its applicability to plants that are difficult to root.
Overall, the manuscript is clearly written. Also, I liked the pictures the authors included; they helped me visualize the different steps of the procedures and the results. There are, however, some changes that would improve the manuscript. One such change is in the statistical analysis. In the manuscript, each explant is considered a replication. However, in most cases, the explants are pseudoreplications. For example, in the experiment involving testing different BAP concentrations, five jars were used for each species and BAP concentration and three explants per jar. The explants within the same jar are not independent; the growth of one may affect the other. Consequently, the true replication is the jar rather than the explant. The correct statistical analysis would involve calculating the average of the three explants per jar and using each average as one of five replications. For the experiment on rooting, all the explants were in one bioreactor; it is too late to correct for pseudo-replications at this point. However, the manuscript mentioned that another trial was conducted and not reported. Assuming the results of this trial were similar to those reported, including them would strengthen the manuscript.
Other minor comments and suggestions related to typos, grammar, references, additional information, and discrepancies between statements in the text and results in the tables are indicated in the attached file.
Comments for author File: 
Comments.pdf
The quality of the English language is good except for some typos and minor grammatical errors.
Author Response
Answer
In the experiment involving testing different BAP concentrations, the explants were considered as single replicates because they were well spaced inside the jar and the growth of one did not influence the development of the other, probably due to the duration of the test (four weeks). Furthermore, explants from the same treatment were quite homogeneous.
For the rooting experiment, since the two trials (September 2022 and July 2023) gave the same results, we decided to include only the results of one of the two trials.
The corrections made to the text were made on a template provided by the editor, while the answers to the other questions are reported on the file sent by the reviewer 2
In the article sent to the editor, corrections are underlined in purple
Author Response File: Author Response.pdf
