A Sustainable Approach to In Vitro Propagation and Conservation of Salvia dominica L.: A Wild Medicinal Plant from Jordan
and Firas Al-Zyoud 6Round 1
Reviewer 1 Report
The work by Al-Qudah et al., set a successful protocol for in vitro shoot proliferation, rooting, acclimatization, and conservation of via vitrification procedure in S. dominca. And this protocol can be used for conserve valuable genetic resources for the long term with high regrowth percentages. The report is well written and condensed, as well as technically appropriate for Sustainability-MDPI. However, before being able to recommend acceptance, I invite authors to address the following amendments.
-In line no.2, 20, 21, 34, 35, 38, 61, 64, 65, 67, 94, 99, 108, 178, 183, 186, 192, 208, 209, 214, 216, 220, 227, 298, 299: "in vitro" it should be italic "in vitro ", please correct it.
-In line no. 39: "Salvia dominica " it should be italic "Salvia dominica ", please correct it.
-In line no. 55 and 65: Please remove the space between "the" and "Salvia species"; and between "on" and "Salvia species ", please correct it.
In line no.81, 174: "In Vitro " it should be "in vitro ", please correct it.
In line no. 249: "Commiphora gileadensis " it should be "C. gileadensis ", please correct it.
Author Response
Dear respected reviewer
Many thanks for your valuable comments that have enriched our research
Reviewer 1:
1- All the "in vitro" phrases were rewritten in italics "in vitro" throughout the manuscript.
2- In line no. 39: "Salvia dominica " was rewritten in italic "Salvia dominica ".
3- The extra spaces were removed in line no. 55 and 65; between "the" and "Salvia species"; and between "on" and "Salvia species "
4- - The word "In Vitro" was corrected to "in vitro" in lines 81, and 174.
5- The words "Commiphora gileadensis" were corrected to "C. gileadensis " in line no. 249.
Reviewer 2 Report
Comments for author File: 
Comments.pdf
the English Grammar should be checked across the manuscript.
Author Response
Dear respected reviewer
Many thanks for your valuable comments that have enriched our research
Reviwer 2:
1- Abstract: Only the best findings and best treatments were mentioned in the abstract as following:
" A full protocol was achieved for shoot proliferation with 6.3 shoots/explant using 1.2 mg L-1 of Thidiazuron (TDZ), while rooting was achieved at 1.5 mg L-1 of NAA with 6.6 functional roots/ explant. Acclimatization was completely successful for the rooted plants. The highest callus production with 5.81 g/calli was achieved using 1.5 mg L-1 of Benzylaminopurine (BAP). Cryopreservation of S. dominica calli was successfully achieved when a pure plant vitrification solution (PVS2) was used to dehydrate calli for 20 min. after immersion in the loading solution for 20 min. with 76.6% regrowth percentage. "
2- The sentence. “Was Optimized” were replaced by a more suitable sentence (A full protocol was achieved) for describing this result – line 26-28, also throughout the manuscript.
3- L45: S. dominica L. was corrected using the full name (Salvia dominica L.) because it was mentioned in first time in the introduction.
4- L103: the unit was corrected from mg.L-1” to “mg L-1” to meet the journal regulation throughout the manuscript.
5- Table 1 was corrected to be a trilinear table. The BAP, Kinein, and TDZ were removed forward of concentrations. An additional column was added as per your suggestion.
6- Table 2 and Table 3 were also corrected as the same as the above Table 1.
7- In Figure 3, the vertical axis titles and horizontal axis headings were provided as you have suggested. Also, the ANOVAs were conducted to test the treatment effects.
8- The vital findings were added to the conclusion (The highest shoot (6.3) and roots (6.6) numbers were obtained using 1.2 mg L-1 of TDZ and 1.5 mg L-1 of NAA; respectively. The rooted plants achieved a full acclimatization process. Also, callus were successfully produced with 5.81 g/calli using BAP at 1.5 mg L-1. On the other hand, the highest regrowth 76.6% was achieved after cryopreservation of S. dominica calli using 0.4 M sucrose + 2M glycerol +HF media as a loading solution for 20 min and 100% PVS2 for 20 min before immersion in the liquid nitrogen.).
9- All sentences were checked for grammar in the whole manuscript.
Reviewer 3 Report
This is an interesting work to establish method for in situ preservation of Salvia plant. Current study presents a systematic method to carry out cryopreservation and clonal propagation in the less studied plant Salvia, which has medicinal value.
There are a few corrections that needed to be made before publication.
1, Line 114, its plastic bag not plastic pages.
2, line 158, calculated spelling should be corrected.
3, Line 220, Figure 1 is distorted, image should be replaced with better quality.
Spelling check is required
Author Response
Dear respected reviewer
Many thanks for your valuable comments that have enriched our research
Reviewer 3
- Line 114, (plastic pages) was corrected to plastic bag.
- line 158, calculated spelling was corrected.
- Line 220, Figure 1 was replaced with better quality images.
- Spelling and grammar were checked throughout the whole manuscript.
Reviewer 4 Report
Comments
The authors studied the in vitro propagation and conservation of Salvia dominica through cryopreservation. There are certain issues associated with the manuscript (MS) that the authors should address before the MS is accepted for publication.
1. The authors may explain why only axillary buds were used as explants, not other parts of the plants. Also, the authors may shed light on why mother stock was initiated using only GA3 and at a particular concentration of 0.5 mg/l.
2. Line-199. Have the authors tried different explants? In the material and methods parts, in vitro shoots with two nodes were used. The authors should show the data with different explants in the MS.
3. Table 1 incorporated callus diameter data, while there are no explanations or mention of the callus formation in the in vitro shoot proliferation section of the MS. The authors should incorporate that part in the results part of the MS. If the callus formation was observed during the shoot proliferation stage, what was need to conduct a new experiment for callus induction for the cryopreservation process?
4. Have the authors tried the derivation of plants from callus after cryopreservation? This stage is crucial for the success of the whole experiment for the conservation of the plants for long-term purposes. Plant generation from callus is always challenging for many plants, requiring a detailed investigation. It will be more difficult for cryopreserved callus. The authors should explain this.
5. The authors are also requested to add more to the discussion part of the MS. There seems to be a lack of reporting of past similar works and critical discussion of the present findings in lieu of the previous findings.
6. The authors failed to study the genetic homogeneity of the in vitro propagated plants. It seemed callus formation was observed during shoot proliferation, so it becomes more important to check the genetic uniformity of the regenerated plants as there was a high chance of occurrence of somaclonal variation. The authors should reply with a solid response in this regard.
7. There are some minor grammatical errors, which the authors may check the whole MS and make necessary corrections. Unless the above-mentioned issues are not addressed, the paper cannot be accepted for publication.
some minor grammatical errors which the authors can correct.
Author Response
Dear respected reviewer
Many thanks for your valuable comments that have enriched our research
Reviwer 4
1- Comment : The authors may explain why only axillary buds were used as explants, not other parts of the plants. Also, the authors may shed light on why mother stock was initiated using only GA3 and at a particular concentration of 0.5 mg L-1.
Reply:
In this study, axillary buds were used as explants to initiate in vitro cultures. This is because they gave a good development under in vitro conditions rather than seeds that were suffering from germination problems. Previous literature reported that Salvia species suffer from seed dormancy [38-40]. Furthermore, most of the previous studies documented the use of axillary buds to initiate the in vitro culture [41-43].
The axillary buds are an asexual propagation method that gives genetically identical plants to the mother plants rather than seeds [41; 43]. According to our objective, we tried to produce a sustainable protocol to keep this valuable plant. Also, to apply this protocol in the future in order to return this valuable plant to its environment after the acclimatization process with fewer variations in the produced plants. The mother stock plants were initiated in our study using GA3 as a growth regulator at different concentrations (0.1, 1.0, and 0.5 mg L-1; (data not shown)) and 0.5 mg L-1 was the only concentration that gave good results for axillary bud development. The gibberellic acid (GA3) was documented in the previous literature to induce growth and for breaking buds dormancy [38; 44].
This reply was documented in manuscript in the result section (3.1. Plant Sterilization and in vitro Establishment)
2- Comment 2: In the materials and method section 2.2.
Only axillary buds were used as explants for in vitro initiation, and these axillary buds have 2 nodes at least to initiate the shoot development from those nods.
3- Comment 3: Table 1 incorporated callus diameter data, while there are no explanations or mention of the callus formation in the in vitro shoot proliferation section of the MS. The authors should incorporate that part in the results part of the MS. If the callus formation was observed during the shoot proliferation stage, what was need to conduct a new experiment for callus induction for the cryopreservation process?
Reply:
In Table 1; the callusing in the base of the in vitro shoot propagation is due to the presence of cytokinin in the media. This callus usually is not friable (compact and brown) and is considered not an advantage. In our MS a small and neglected callus was observed on the in vitro shoot base. Using TDZ at higher concentrations caused an increase in the callus diameter at the base of the in vitro shoot. This callus will be removed if present when shoots are in vitro rooted.
The explanations of the callus formation in the in vitro shoot proliferation section were added to the MS in the section: 3.2. In vitro S. dominica proliferation, rooting, and acclimatization; as follows:
"In our study, a small and neglected callus was observed on the in vitro shoot base. Except for using TDZ at high concentrations where the callus diameter at the base of the in vitro shoots reached up to 2.18 cm at 2.0 mg L-1 TDZ. Similarly; TDZ at 2.0 mg L-1 produced a higher callus diameter (8.8 mm) at the base of T. polium in vitro shoots [41]."
A new experiment for callus induction is necessary to produce healthy and friable calli that can be multiplied and can be used for further experiments such as for the cryopreservation process.
4- Have the authors tried the derivation of plants from callus after cryopreservation? This stage is crucial for the success of the whole experiment for the conservation of the plants for long-term purposes. Plant generation from callus is always challenging for many plants, requiring a detailed investigation. It will be more difficult for cryopreserved callus. The authors should explain this.
Reply:
Plant regeneration from callus was not done in this study. This may be used in further studies in the future. The callus is considered a valuable genetic plant material of undifferentiated tissue that has all the genetic information [64]. Previous literature documented the use of the callus for cryopreservation methods. Callus cryopresrvion was used in some studies without regeneration experiments [65-66]. The ability of the callus regeneration is due to the totipotency potential in the plant cell to give new plants through direct and indirect embryogenesis [64].
So, if we have a plant callus material we have all the genetic information needed in the plant. Also, we can use callus to do further regeneration studies on it.
5- Reply: The discussion part of the MS was enriched to be more informative and describe the present findings.
6- Reply: In our study, the mother stock in vitro shoots were developed from axillary buds in section 3.1. In the in vitro shoot experiments in section 3.2 (Table 1); no regeneration was observed from the callus. The friable and compacted callus was formed on the base of the in vitro shoots and was neglected. Calli was established directly from leaf segments in section 3.3.
In the plant tissue culture studies, the genetic homogeneity of the in vitro propagated plants in most cases is still constant. The somaclonal variation in the plant tissue culture is affected by the growth regulators used and the direct and indirect regeneration [67-68]. However, most reports found a low percentage of somaclonal variation occurrence in tissue-cultured plants [69-70]. In this study, no genetic stability was performed. However, some literature has documented the genetic stability after cryopreservation. For example; no genetic variations were detected in the cryopreserved shoots of Thymbra spicata [16], Rubus germplasm [71] and wasabi shoot tips [72] before and after cryopreservation. This study also may open the gate to further research depending on the current data with other details and new approaches.
7- Grammatical errors and typos were corrected throughout the whole MS
Reply:
In Table 1; the callusing in the base of the in vitro shoot propagation is due to the presence of cytokinin in the media. This callus usually is not friable (compact and brown) and is considered not an advantage. In our MS a small and neglected callus was observed on the in vitro shoot base. Using TDZ at higher concentrations caused an increase in the callus diameter at the base of the in vitro shoot. This callus will be removed if present when shoots are in vitro rooted.
The explanations of the callus formation in the in vitro shoot proliferation section were added to the MS in the section: 3.2. In vitro S. dominica proliferation, rooting, and acclimatization; as follows:
" In our study, a small and neglected callus was observed on the in vitro shoot base. Except for using TDZ at high concentrations where the callus diameter at the base of the in vitro shoots reached up to 2.18 cm at 2.0 mg L-1 TDZ. Similarly; TDZ at 2.0 mg L-1 produced a higher callus diameter (8.8 mm) at the base of T. polium in vitro shoots [35]."
A new experiment for callus induction is necessary to produce healthy and friable calli that can be multiplied and can be used for further experiments such as for the cryopreservation process.
4- Have the authors tried the derivation of plants from callus after cryopreservation? This stage is crucial for the success of the whole experiment for the conservation of the plants for long-term purposes. Plant generation from callus is always challenging for many plants, requiring a detailed investigation. It will be more difficult for cryopreserved callus. The authors should explain this.
Reply:
Plant regeneration from callus was not done in this study. This may be used in further studies in the future. The callus is considered a valuable genetic plant material of undifferentiated tissue that has all the genetic information [58]. Previous literature documented the use of the callus for cryopreservation methods. Callus cryopresrvion was used in some studies without regeneration experiments [59-60]. The ability of the callus regeneration is due to the totipotency potential in the plant cell to give new plants through direct and indirect embryogenesis [58]. So, if we have a plant callus material we have all the genetic information needed in the plant. Also, we can use callus to do further regeneration studies on it.
5- Reply: The discussion part of the MS was enriched to be more informative and describe the present findings.
6- Reply: In our study, the mother stock in vitro shoots were developed from axillary buds in section 3.1. In the in vitro shoot experiments in section 3.2 (Table 1); no regeneration was observed from the callus. The friable and compacted callus was formed on the base of the in vitro shoots and was neglected. Calli was established directly from leaf segments in section 3.3.
In the plant tissue culture studies, the genetic homogeneity of the in vitro propagated plants in most cases is still constant. The somaclonal variation in the plant tissue culture is affected by the growth regulators used and the direct and indirect regeneration [61-62]. However, most reports found a low percentage of somaclonal variation occurrence in tissue-cultured plants [36-64]. In this study, no genetic stability was performed. However, some literature has documented the genetic stability after cryopreservation. For example; no genetic variations were detected in the cryopreserved shoots of Thymbra spicata [12] and Ziziphora tenuior [65] before and after cryopreservation. This study also may open the gate to further research depending on the current data with other details and new approaches.
7- Grammatical errors and typos were corrected throughout the whole MS
Round 2
Reviewer 2 Report
All of my concern have been addressed well. However, I have a minor suggestion: For one sentence, no more than three refences may be enough. In total, no more than 70 references could be better for the manuscript.
Minor editing of English language required. Such as the first sentence in abstract. "Salvia dominica L., as a wild plant, is an important medicinal genetic resource" could be better.
Author Response
Many thanks for your commnets
1- According to your suggestion: for one sentence, no more than three references were addressed. So, the total, number of references were reduced less than 70 to be (66).
2- I have checked all the sentences and English grammer and for the
first sentence in the abstarct I have changed to "Salvia dominica L. is an important wild medicinal plant that grows in Jordan and neighboring countries and has been suffering from many threats in its wild environment."
Author Response File: Author Response.pdf
Reviewer 4 Report
The authors have addressed almost all the issues raised by the reviewer. There is a significant improvement in the MS and the it can be accepted for publication in the journal.
Author Response
Many thanks for your valuable recommendations that have added a significant improvement to our MS
Author Response File: Author Response.pdf
