Nursery Propagation Systems for High-Quality Strawberry (Fragaria × ananassa Duch.) Plug Plant Production from Micropropagated, Soilless-Grown Mother Plants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

The authors are encouraged to revise and adjust the manuscript based on the observations provided throughout the document, particularly regarding the overly extensive writing in sections such as the conclusions, the absence of complementary analyses to better support the results (economic, physiological, or post-harvest), and the need to strengthen the critical discussion of the practical and regulatory implications of the proposed system. A more concise version, with deeper analytical insights and improved structure, would enhance both the scientific impact and practical relevance of the work.

Regards

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please improve fluency by breaking up long sentences. Althougt correct prepositions, verb tenses and articles.

 

Author Response

Reviewer 1

The authors are encouraged to revise and adjust the manuscript based on the observations provided throughout the document, particularly regarding the overly extensive writing in sections such as the conclusions, the absence of complementary analyses to better support the results (economic, physiological, or post-harvest), and the need to strengthen the critical discussion of the practical and regulatory implications of the proposed system. A more concise version, with deeper analytical insights and improved structure, would enhance both the scientific impact and practical relevance of the work.

• The introduction should be restructured to improve its clarity and scientific approach. It is currently long, with confusing wording, unnecessary repetitions and without a clear delimitation of the problem or the objective of the study. It is suggested to divide it into thematic blocks (productive context, phytosanitary limitations, potential of micropropagation and objective of the work) and close with a clear paragraph that formulates the hypothesis or experimental purpose. In addition, give more weight to the agronomic topic.

Abstract, introduction and key words have been totally revised.

• The environmental conditions of the soilless system should be clarified and the agronomic justification for specifically selecting the Dina variety in the evaluation
  “The greenhouse was equipped with an air conditioning system for controlling temperature, sunshine and ventilation. From April to September only the ventilation and the shading system with dark nets were activated. The length of day hours for the requested photoperiod (16 hours) was naturally granted without the need of artificial illumination. The automatic system of ventilation and shading was set with sensors: openings and closings of lateral greenhouse barriers were ensured with temperatures superior to 20-25 °C. This system contributed to maintaining an ambient humidity of 60-70%, so reducing the risk to reach humidity levels that can promote high development of fungal diseases. The greenhouse management was set for assuring the best climatic conditions related to a faster morpho – physiological development for a vegetative fruitfulness of the mother plants.”

 

“As ‘Dina’ was intended for shipment to Southern regions for soilless transplanting by September, we evaluated different nursery fertigation protocols to accelerate runner tip rooting and production.”

• Why was the maximum number of subcultures chosen? Was it based on physiological or productive criteria?

This decision was driven by the aim to establish a stable and efficient multiplication rate, promoting shoot proliferation and reliable morphogenetic responses. According to standard micropropagation protocols, repeated subculturing over several cycles allows for the stabilization of growth patterns and minimizes physiological or developmental anomalies that may occur in earlier stages. Moreover, maintaining the number of subcultures below ten is widely regarded as a precautionary measure to preserve genetic fidelity during clonal propagation, especially when callus formation is avoided in favor of organized shoot development. Eight subcultures represent a balance between achieving the desired multiplication output and minimizing the risk of undesirable genotypic or phenotypic variation, as recognized in micropropagation best practices.

• Please justify the choice of the LSD test versus more robust methods such as Tukey in the presence of multiple

The LSD test was chosen over more conservative post hoc methods such as Tukey’s HSD to enhance sensitivity in detecting treatment differences. Given the limited number of treatments and our focus on subtle genotypic and micropropagation-related differences, LSD provided sufficient statistical power. Sampling from the central plants further reduced variability associated with edge effects, reinforcing the reliability of our comparisons.

• High observed variability (e.g., high standard deviations) should be discussed as a possible limiting factor in the uniformity of propagated material.

The high standard deviation observed in the number of runner tips produced by micropropagated mother plants can be attributed to the intrinsic variability in the stoloniferous behavior of this genotype. This heterogeneity is likely influenced by both physiological factors linked to the stage of acclimatization and environmental microvariations within the cultivation system. Additionally, the asynchronous development of tips and the competition for assimilates between vegetative growth and tips formation in everbearing types may have further contributed to the observed dispersion in the data.

• Although significant differences between genotypes in stoloniferous capacity are highlighted, it is suggested that a multivariate analysis (such as PCA or cluster analysis) be incorporated to identify genetic groupings with similar yield patterns. In addition, it would be valuable to complement yield data with indicators of tip morphological quality to strengthen the commercial applicability of the results.

PCA analyses were performed and these also testified to the greater stoloniferous potential of cv Francesca and Silvia, in contrast to Lauretta, AN,12,13,58 and Dina. Therefore, the stoloniferous capacity of the plants themselves is a cv-dependent parameter.

I would like to point out that, according to our statistics consultant, since we only have 3 variables, it would not be necessary to perform a PCA, and a three-dimensional plot would be sufficient. Therefore, we have added the PCA as requested by the reviewer and the three-dimensional plot (Figure 2 a, b and c).

Reviewer 2 Report

Comments and Suggestions for Authors

This study focuses on the potential of micropropagated strawberry mother plants for enhanced runner and tip development in a soilless system. Despite the topic's relevance to contemporary nursery practices, the manuscript's influence is now limited by a number of methodological and structural flaws. Therefore I could not recommend this paper for the following reasons.

Key Issues:

• The lack of interannual replication in the single-season examination (2022) raises concerns regarding the consistency of the results under various growing circumstances.
  Statistical power is diminished by the low replication (n=4 for mother plants and n=6 for Dina tips), especially when it comes to genotype comparisons. Results would be strengthened by more trials and a larger sample size.
• In line 299: You present only the results for AN12,13,58 are shown prominently, but in the Materials and Methods section is not specified that these metrics are measured only for this genotype.
• It seems as though the fertigation experiment (NPK treatments for Dina tips) is unrelated to the primary research. Either explain how it relates to the propagation system or think about presenting it independently in another paper. The outcomes of the fertigation seem "tacked on" instead of integrated. Either clearly connect it to the objectives of the propagation system (e.g., tip quality for field performance), or take it out to concentrate on the main comparison between mother plants that are micropropagated and those that are in vivo.
• Fruit performance: Although the study concentrates on replication efficiency, it leaves out information on the fruit yield and quality of the plants that were propagated, which is an important statistic for producers.
• Economic analysis: Although fewer mother plants are produced by the approach, infrastructure expenses (soilless setup, micropropagation lab) are mentioned but not measured. Applicability would be strengthened by a cost-benefit analysis compared to conventional techniques.

Specific comments:

Material and methods

Line 152 : what are those protocols?

Line 164: You should state the composition of the MS medium

Line 172: specify the amount of the added cytokine

Lines 183-185: explain why these data were not collected

Lines 202-203: you said two dates but you mention one date. Please check your sentence.

Line 229:explain why this experiment was performed only for Dina cultivar

Lines 261-274: should be in a paragraph and not in bullets

Line 277: explain what mean L, C and h°

Line 295: Specify the type of ANOVA (one-way or two-way)

Results and discussion

Line 299: why have you presented only results for AN12,13,58 it is not mentioned in the material and methods section?

Lines 301-302: this result does not exist in Table 1

Table 4: what ° means in 10.42±0.41° it should be a letter here

Line 473: what does this mean? What is the best fertigation treatment? You did not clearly describe the results

 

 

 

 

 

Author Response

Reviewer 2

This study focuses on the potential of micropropagated strawberry mother plants for enhanced runner and tip development in a soilless system. Despite the topic's relevance to contemporary nursery practices, the manuscript's influence is now limited by a number of methodological and structural flaws. Therefore, I could not recommend this paper for the following reasons.

Key Issues:

• The lack of interannual replication in the single-season examination (2022) raises concerns regarding the consistency of the results under various growing circumstances.
  Statistical power is diminished by the low replication (n=4 for mother plants and n=6 for Dina tips), especially when it comes to genotype comparisons. Results would be strengthened by more trials and a larger sample size.
  As explained in paragraph “statistical analysis”, we used to have only 4 samples for each bags considering the central part of the bag for not having the edge effects for the analyzed treatments. Moreover, knowing that, we used a more precise statistical tool such as LSD test because differences between the cv was monitored. For the rooted tips we mentioned the fact that 6 samples are poorly representative, especially considering the analysis with WINRhizo. However, it is possible to consider this as a starting point for another research.
• In line 299: You present only the results for AN12,13,58 are shown prominently, but in the Materials and Methods section is not specified that these metrics are measured only for this genotype.
  We correct the phrase in material and method where we presented the selection AN12,13,58. Moreover, reading the paragraph “Production of micropropagated and in vivo mother plants”, we explained the vegetative propagation for the obtainment of the type A plants.
  
  
• It seems as though the fertigation experiment (NPK treatments for Dina tips) is unrelated to the primary research. Either explain how it relates to the propagation system or think about presenting it independently in another paper. The outcomes of the fertigation seem "tacked on" instead of integrated. Either clearly connect it to the objectives of the propagation system (e.g., tip quality for field performance), or take it out to concentrate on the main comparison between mother plants that are micropropagated and those that are in vivo.
  
  
• Our objective is to describe the differential response of runner tips to fertigation, with the goal of optimizing nursery plant production. By using micropropagated mother plants, we obtained a higher number of runner tips, and our first priority is to demonstrate the high quality of this new plant material. The experiment conducted exclusively with the ‘Dina’ cultivar provides a preliminary indication of the quality of plants obtained through this propagation method, and for this reason, we consider it a valuable contribution to complete the present manuscript. Additionally, it is important to assess early growth responses during the initial rooting phase of runner tips, taking into account specific fertigation programs. The experience gained with cv. Dina is easily transferable to other cultivars as well. Broader experiments aimed at evaluating the performance of plants derived from rooted runner tips in open-field fruit production are currently underway and will be the subject of a separate publication.

 

• Fruit performance: Although the study concentrates on replication efficiency, it leaves out information on the fruit yield and quality of the plants that were propagated, which is an important statistic for producers.
  This study is specifically aimed at optimizing the cultivation technique for micropropagated mother plants, taking into account the effects of different genotypes and planting times on the efficient production of high-quality stolon chains.

The evaluation of the productive performance of the plants obtained represents a separate line of research that has already been initiated and will be the subject of future publication. However, preliminary studies have already shown that plants produced through this method do not differ from those derived from conventionally propagated mother plants (see citation no. 21).

• Economic analysis: Although fewer mother plants are produced by the approach, infrastructure expenses (soilless setup, micropropagation lab) are mentioned but not measured. Applicability would be strengthened by a cost-benefit analysis compared to conventional techniques.
  Our focus is on agronomic aspects rather than economic ones, with the aim of highlighting the potential of micropropagation protocols and the nursery performance of the resulting mother plants.

Production costs are closely linked to the type of nursery infrastructure and should be further analyzed by considering the expenses associated with the different facilities and equipment required for the two propagation techniques. The proposed system undoubtedly involves higher structural costs (micropropagation laboratory and greenhouses), but it offers significantly greater efficiency in stolon chain production compared to conventional nursery-grown plants—along with a much higher level of sanitary quality, which is now a critical requirement in the strawberry nursery market.

The calculations regarding stolon chain production per square meter were included specifically to demonstrate the high efficiency of this system, particularly when using micropropagated mother plants. This information is essential for any meaningful comparison of the economic costs of the proposed system versus traditional propagation methods.

 

Specific comments:

Material and methods

Line 152 : what are those protocols? The protocol cited with reference 19 is the same explained from lines 157 – 171.

Line 164: You should state the composition of the MS medium
MS medium powder (Duchefa Biochemie, The Netherlands) was used according to the manufacturer’s instructions – now better specified.

Line 172: specify the amount of the added cytokine (0.25 mg/l, written)

Lines 183-185: explain why these data were not collected “Data on the response of the different genotypes to the proliferation, rooting and acclimatization phases of the micropropagation cycle were not collected because the primary focus was to evaluate the propagation performance of the mother plants under in vivo conditions allowing us to concentrate here on nursery performance for practical applications” – now better specified.

Lines 202-203: you said two dates but you mention one date. Please check your sentence. “This operation was conducted on 13/04/2022 for the cultivars Francesca, Lauretta, Silvia and AN12,13,58.” – now better specified.

Line 229: explain why this experiment was performed only for Dina cultivar
“As ‘Dina’ was intended for shipment to Southern regions for soilless transplanting by September, we evaluated different nursery fertigation protocols to accelerate runner tip rooting and production.” – now better specified.

Lines 261-274: should be in a paragraph and not in bullets
We have eliminated the bullets and re-written the paragraph.

Line 277: explain what mean L, C and h°
We have explained these values on “2.6 Data of qualitative response for Dina Nursery tips production”

Line 295: Specify the type of ANOVA (one-way or two-way)
Specified (one-way ANOVA), – now better specified.

Results and discussion

Line 299: why have you presented only results for AN12,13,58 it is not mentioned in the material and methods section?
Yes is mentioned, we have corrected the phrase in paragraph “2.1. Strawberry mother plant: cultivars and selection” as suggested by reviewer 1. – now better specified.

Lines 301-302: this result does not exist in Table 1
“In fact, before harvesting the in vitro mother plants counted 13.42±6.19 runners per plant against 4.06±1.24 of the in vivo one.” Referred to Table 2.

Table 4: what ° means in 10.42±0.41° it should be a letter here
It’s a typo. – now corrected.

Line 473: what does this mean? What is the best fertigation treatment? You did not clearly describe the results
The paragraph is now more properly written.

Reviewer 3 Report

Comments and Suggestions for Authors

This research work has important theoretical and practical significance, especially for the development of related industries with significant reference value. The overall quality of the article is good. After reviewing, please answer the following questions.
1. The abstract can be further refined.
2. In order to compare the advantages and disadvantages of microproduced technology, the author selected multiple morphological indicators for evaluation. ① Why choose these indicators instead of indicators for yield and quality? ② Are these indicators contributing equally to the evaluation? Which ones are more crucial? So that producers can better and more easily apply the results of this article to guide their strawberry production.
3. Does' it was estimated 'mentioned by L376 mean estimated, not measured?
4. The conclusion can also be further refined.

Author Response

Reviewer 3

This research work has important theoretical and practical significance, especially for the development of related industries with significant reference value. The overall quality of the article is good. After reviewing, please answer the following questions.
1. The abstract can be further refined.
We worked on it.

2. 
   In order to compare the advantages and disadvantages of microproduced technology, the author selected multiple morphological indicators for evaluation.
   ① Why choose these indicators instead of indicators for yield and quality?

Evaluating the quality of mother plants obtained through micropropagation is essential to ensure the efficiency of clonal multiplication and the production of uniform, vigorous, virus – free and physiologically active plant material. To this end, the use of specific parameters allows for an objective assessment of the vegetative vigor of the plants. The parameters selected are the commonly used and informative indicators:

1. Number of runners, number of runner tips and stolon length

• These are direct indicators of the plant’s reproductive potential, particularly relevant in stoloniferous species such as strawberry.
• Stolon production is regulated by hormonal factors (e.g., auxins and gibberellins) and is often correlated with optimal physiological status.
• Greater stolon length and branching reflect high meristematic activity and efficient resource allocation, both of which indicate overall plant health and juvenility thanks of in vitro propagation.
• Moreover, the development of complex stoloniferous chains (i.e., multiple nodes and clones) is agronomically advantageous, reducing both time and propagation costs in a strawberry nursery life cycle.

2. Fresh and dry weight of leaves and root systems in rooted tips

• Fresh weight indicates current vegetative development and tissue hydration, which is relevant for assessing transplant viability.
• Dry weight reflects the actual accumulation of structural and metabolic biomass and is a more stable indicator of active growth and photosynthetic efficiency.
• Root biomass is directly related to nutrient and water uptake efficiency and determines the plant's stability after transplanting.

We considered together, these parameters allow for an objective evaluation of the quality of micropropagated material, both in the mother plants and their clonal progeny. This permit to have a prediction of agronomic performance following transplant. This is going be a scientific basis for observing if the in vitro protocols optimize the nursery response. In summary, these morphophysiological traits are easy to measure, highly informative, and closely associated with the agronomic and commercial performance of tissue-cultured plants.

A synthesis of these motivations have been added.

② Are these indicators contributing equally to the evaluation? Which ones are more crucial? So that producers can better and more easily apply the results of this article to guide their strawberry production.

From a producers’ perspective, the runners emission and the higher rate of runner tips of the micropropagated mother plant are the most critical parameter for assessing the technical and economic competitiveness of a micro-plants system. While fresh and dry biomass are relevant indicators of fresh rooted plants quality. They are secondary in terms of direct commercial impact because they reflect the final performance of individual plants rather than the propagation system’s overall capacity to generate value.

A synthesis of these motivations have been added.

3. 
   Does' it was estimated 'mentioned by L376 mean estimated, not measured?

The data collected 60 days after transplanting confirmed the potential of in vitro-derived plants in a soilless cultivation system. However, the experimental setup involved only four bags per variety, totaling 24 mother plants within a 4-meter section of the greenhouse. For the statistical analysis we considered only the 4 central mother plants per bag. To provide relevant insights for nurseries, it is important to scale the results according to practical production targets—such as the estimated need for 100,000 rooted tips. Communicating results based solely on a limited number of plants may be less impactful, as growers are primarily interested in performance at a commercial scale rather than research-scale observations. This becomes a way to communicate research at the corporate scale.

4. The conclusion can also be further refined.
   We worked on it.
   
   

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors

The new version of manuscript its ok.

regards

Author Response

We thank reviwer 1 for the final positive evalution of our manuscript.  

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors' Responses to Reviewer's Comments are not well presented: authors sometimes did not refer to the changes in the text, and when they did, the line numbers mentioned did not correspond to either the old version or the revised version whixh make the revision more difficult.

This manuscript still have issus depite the revision:

• The lack of interannual replication in the single-season examination (2022) raises concerns regarding the consistency of the results under various growing circumstances.
• Statistical power is diminished by the low replication (n=4 for mother plants and n=6 for Dina tips), especially when it comes to genotype comparisons. Results would be strengthened by more trials and a larger sample size.
• It seems as though the fertigation experiment (NPK treatments for Dina tips) is unrelated to the primary research. Either explain how it relates to the propagation system or think about presenting it independently in another paper. The outcomes of the fertigation seem "tacked on" instead of integrated. Either clearly connect it to the objectives of the propagation system (e.g., tip quality for field performance), or take it out to concentrate on the main comparison between mother plants that are micropropagated and those that are in vivo.

And the answers given by the authors  for 2) and 3) are not convincing.

• My comment: Line 152 : what are those protocols? 

Your answer:The protocol cited with reference 19 is the same explained from lines 157 – 171.

My new comment: I could not found the protocol and the number lines correspond to which version.

• My comment: Line 164: You should state the composition of the MS medium
  Your answer: MS medium powder (Duchefa Biochemie, The Netherlands) was used according to the manufacturer’s instructions – now better specified.

My new comment: you still did not specify the composition of the MS medium

 

• In figure 2: letters are not well aligned, the (a) is trunked, the (b) is missed and it is written ()- instead.

 

Author Response

REVIEW 2: The Authors' Responses to Reviewer's Comments are not well presented: authors sometimes did not refer to the changes in the text, and when they did, the line numbers mentioned did not correspond to either the old version or the revised version which make the revision more difficult.

This manuscript still have issues depite the revision:

• The lack of interannual replication in the single-season examination (2022) raises concerns regarding the consistency of the results under various growing circumstances.
  The study was conducted over a single growing season, which represents a limitation in terms of long-term reproducibility and we acknowledge this limitation and we have clarified it in the revised manuscript. The experiment was designed as a focused trial conducted in a controlled greenhouse, that helped to reduce external variability. In fact, these conditions helped also for the evaluation of the early propagation performance of micropropagated strawberry plants under standardized environmental and nutritional levels. The current study thus provides useful preliminary insights and a methodological foundation for future multi-season or field-scale investigations.
  The use of 16 plants per cultivar (4 plants x 4 bags) reflects a common practice to study in vivo plants from micropropagation, especially when working with limited high-value plant material in early-stage trials.
  Moreover, in order to maximize the scientific value of the single-season study, we chose to expand the investigation to include the production of rooted fresh plant of Dina cultivar. This choice was motivated by the commercial interest in Dina, particularly due to its propagation capacity and the speed at which daughter plants can be obtained—two key factors in nursery productivity and protocol efficiency.
• Statistical power is diminished by the low replication (n=4 for mother plants and n=6 for Dina tips), especially when it comes to genotype comparisons. Results would be strengthened by more trials and a larger sample size.

We agree that statistical power is affected by the limited replication (n = 4 for mother plants, n = 6 for Dina tips). This study represents a first step in the evaluation of propagation performance under controlled conditions, and was designed to generate preliminary data for protocol development. As above, the use of controlled greenhouse conditions minimized environmental variability, allowing for meaningful biological interpretations despite the low sample size. We interpreted the results conservatively (LSD test instead of Tukey Test) and from these findings we observed valuable preliminary evidences and consistent trend (see paragraph 3.1) to justify and guide further investigations.

• It seems as though the fertigation experiment (NPK treatments for Dina tips) is unrelated to the primary research. Either explain how it relates to the propagation system or think about presenting it independently in another paper. The outcomes of the fertigation seem "tacked on" instead of integrated. Either clearly connect it to the objectives of the propagation system (e.g., tip quality for field performance), or take it out to concentrate on the main comparison between mother plants that are micropropagated and those that are in vivo.

We appreciate the reviewer’s observation regarding the apparent separation between the main propagation system comparison and the fertigation experiment conducted on rooted tips of cv ‘Dina’. However, we would like to clarify that the two components are conceptually and practically connected within the broader framework of evaluating the propagation system in strawberry. The stolon production capacity of mother plants was assessed to compare propagation potential between in vitro genotype. For cv. ‘Dina’, which could be rapidly shipped for an autumn transplanting, we extended the evaluation to the quality of the resulting tips, focusing on their physiological and morphological traits in response to different NPK fertigation treatments. This additional analysis allowed us to better characterize the performance and quality of the propagules derived from Dina in vitro mother plant, thus integrating both the quantitative (stolon number) and qualitative (tip development) aspects of the propagation process. Our intention was not to introduce an unrelated experiment, but rather to provide a more complete picture of the propagation potential and downstream performance of the produced material, particularly for a cultivar with high commercial interest. To address the reviewer’s concern, we have revised the introduction (see lines 85 – 94) to explicitly state the rationale behind including the fertigation experiment, and its relevance to the evaluation of propagation system efficiency.

• And the answers given by the authors  for 2) and 3) are not convincing.

2) see lines 86 - 96, 188 - 198, 200 – 204

3) As the last time, we confirm that this decision was driven by the aim to establish a stable and efficient multiplication rate, promoting shoot proliferation and reliable morphogenetic responses. According to standard micropropagation protocols, repeated subculturing over several cycles allows for the stabilization of growth patterns and minimizes physiological or developmental anomalies that may occur in earlier stages. Moreover, maintaining the number of subcultures below ten is widely regarded as a precautionary measure to preserve genetic fidelity during clonal propagation, especially when callus formation is avoided in favor of organized shoot development.

In Italy, official guidelines for the commercial certification of micropropagated strawberry plants generally limit the number of in vitro subcultures to a maximum of five, in order to reduce the risk of somaclonal variation and ensure traceability. However, this threshold, while appropriate for certified plant production, can represent a limiting factor in research contexts, where more extended subculturing is sometimes necessary to achieve in vitro stability and consistent proliferation rates. In the present study, we exceeded the fifth subculture cycle for experimental purposes, and observed stable morphogenetic behavior, high-quality shoot formation, and no detectable phenotypic or productivity-related abnormalities. These results confirm that, under controlled conditions and with strict avoidance of callus phases, clonal fidelity and performance can be maintained even beyond five subcultures. This is another advantage of the proposed protocol.

• My comment: Line 152 : what are those protocols? 

Your answer: The protocol cited with reference 19 is the same explained from lines 157 – 171.

My new comment: I could not found the protocol and the number lines correspond to which version.

Now, in the new version of the article, we have changed the sentence setting. We now start directly with the description of the sterilization procedure, while still remaining the citation to Article 19 (lines 108 – 112).

• My comment: Line 164: You should state the composition of the MS medium
  Your answer: MS medium powder (Duchefa Biochemie, The Netherlands) was used according to the manufacturer’s instructions – now better specified.

My new comment: you still did not specify the composition of the MS medium

We have now better specified the composition of MS medium, according to the recipe adopted by Duchefa Biochemie (lines 115-120).

In figure 2: letters are not well aligned, the (a) is trunked, the (b) is missed and it is written ()- instead.
It’s a typo – now correct.