Plant Production and Leaf Anatomy of Mertensia maritima (L.) Gray: Comparison of In Vitro Culture Methods to Improve Acclimatization

Round 1

Reviewer 1 Report

The manuscript titled „Plant production and leaf anatomy of Mertensia maritima (L.) Gray: comparison of in vitro culture methods to improve acclimatization” is well written. The subject of the study is very interesting due to the rare plant species and the limitted studies on its micropropagation.

Some points that shall be amended:

Introduction:

The expression „the morpho-physiological characters” should be changed into morpho-anatomical …” as no phisiological processes were tracked during experiments.

The latin expressions „in vitro” and „in vivo” shall be written with italics.

Materials and methods:

The value of the light irradiances at jar level is quite unusuall and very high (209 ± 5 μmol m−2 s −1). Can authors precise the distance between lamps and plant material in the jar?

The heading of the subsection 2.6 „Leaf morphological and anatomical analyses” could be changed into „Leaf morphological and anatomical examination”

Results:

The authors wrote: „After four weeks of culture, the shoot clusters from TIS showed a significantly higher biomass (both fresh and dry)”. The results shown in Table 1 do not show a significant difference in the biomass production between jars and TIS, even though the statistical analysis confirmed it, but it has to be admitted that there are large differences in the morphology of cultured plants. So, it would be better to rewrite this paragraph with all the detailes on the numer and size of shoots and leaves. The one missing parameter is the length of the axillary shoots. It should be entered in the Table 1.

The authors wrote „The shoot multiplication is significantly greater in the culture in semi-solid substrate than in the liquid one, while plant biomass and leaf parameters were higher in the liquid culture.” – this issue should be discussed in more detail, as the aim of the protection of endangered species is their multiplication through micropropagation. The more so, it was presented as one of the main conclusions.

Author Response

Response to Reviewer 1 Comments

Point 1: The expression „the morpho-physiological characters” should be changed into morpho-anatomical …” as no phisiological processes were tracked during experiments.. 


Response 1: the text has been changed

Point 2: The latin expressions „in vitro” and „in vivo” shall be written with italics.

Response 2: the Latin expressions have been written in italics

Point 3: The value of the light irradiances at jar level is quite unusuall and very high (209 ± 5 μmol m−2 s −1). Can authors precise the distance between lamps and plant material in the jar?

Response 3: the distance between lamps and plant material in the jar was 42 cm. The value of light radiation at the level of the jar was measured at the level of the lid of the jar.

Point 4: The heading of the subsection 2.6 „Leaf morphological and anatomical analyses” could be changed into „Leaf morphological and anatomical examination”

Response 4: the text has been changed

Point 5: The authors wrote: „After four weeks of culture, the shoot clusters from TIS showed a significantly higher biomass (both fresh and dry)”. The results shown in Table 1 do not show a significant difference in the biomass production between jars and TIS, even though the statistical analysis confirmed it, but it has to be admitted that there are large differences in the morphology of cultured plants. So, it would be better to rewrite this paragraph with all the detailes on the numer and size of shoots and leaves. The one missing parameter is the length of the axillary shoots. It should be entered in the Table 1.

Response 5: We thank the referee for having noticed the mistake: the sentence has been changed. The length of auxillary shoot was not measured.

Point 6: The authors wrote „The shoot multiplication is significantly greater in the culture in semi-solid substrate than in the liquid one, while plant biomass and leaf parameters were higher in the liquid culture.” – this issue should be discussed in more detail, as the aim of the protection of endangered species is their multiplication through micropropagation. The more so, it was presented as one of the main conclusions.

Response 6: the discussion was expanded (from 391 line to 409 line).  the main aim of the study was to identify the best protocol to multiply and obtain a high number of M. Maritima plants set for nursery production. Obviously the method is also applicable for the reintroduction programs of this species in nature.

Author Response File: Author Response.pdf

Reviewer 2 Report

Thank you very much for the invitation to review the manuscript entitled "Plant Production and Leaf Anatomy of Mertensia maritima (L.) Gray: Comparison of in vitro Culture Methods to Improve Acclimatization  (1202681, Horticulturae). This is an interesting manuscript but I have some comments that I would like to address to the Authors. The novelty of the manucsript is the use of the temporary immersion system for the multiplication of the Mertensia maritima.

1. Firsty, I have a question regarding to the selection of an explants. Why did the Authors choose microcuttings for the study?
2. Semi-solid medium with 0.75% agar - in my opinion this medium was solid but not semi-solid medium.
3. Line 90: "6-benzyl-aminopurine (BA)" - please correct abbreviation to "BAP". Abbreviation BA stands for benzyladenine.
4. In the Statistical analysis the Authors did not describe how the normality of the data was checked.
5. Why did the Authors the shoot multiplication and root induction present from one representative trial (lines 186-187) but not as the average of the three trials.
6. Lines 195-198: "Nevertheless, plants in TIS showed the lowest number of shoots per cluster and, some hyperhydricity symptoms were observed, especially at the base of the clusters (Fig. 2). For these last two reasons, the micropropagation in jar system was chosen to produce M. maritima shoots to be used for the subsequent experiments of rooting induction." What was the percentage of hyperhydricity shoots? The hyperhydricity shoots are not visible on the Figure 2. In my opinion, the shoots micropropagated in temporary immersion system (Plantform) were better quality and should be used also for the rooting. Moreover, the above sentences (lines 195-198) contradict the results presented in the Discussion (lines 329-331: "The liquid culture in temporary immersion system (TIS) improved the dimension of micropropagated shoots, increased the biomass (both fresh and dry), and allowed to obtain longer leaves with longer and wider leaf blades, in comparison with the common jar system with agarized semisolid substrate."
7. Line 327-328: "The shoot number per cluster observed in semisolid culture was comparable to that registered to Park et al. [5],...." but Park et al. [5] and Song et al. [6] obtained a much higher multiplication rate after using nodes as explants. Therefore, the use of the temporary immersion system for the multiplication of Mertensia maritima in present study did not improve the micropropagation of these species but it affect the shoot rooting (although, the results also similar to those obtained by Park et al. and Song et al.) and plant acclimatization.
8. The survival rate of the shoots after acclimatization in green house was very low (3-5%) and extremely unsatisfactory, therefore, Mertensia maritima plants can not be obtained on an industrial scale.

Author Response

Response to Reviewer 2 Comments

Point 1: Firsty, I have a question regarding to the selection of an explants. Why did the Authors choose microcuttings for the study?


Response 1: the starting material were mother plants grown in pots. In our experience, the surface of 1 cm long cuttings (microtalee) is easily sterilized

Point 2: Semi-solid medium with 0.75% agar - in my opinion this medium was solid but not semi-solid medium.

Response 2: the substrate is gelatinous and therefore semi-solid

Point 3: Line 90: "6-benzyl-aminopurine (BA)" - please correct abbreviation to "BAP". Abbreviation BA stands for benzyladenine.

Response 3: BA has been changed to BAP

Point 4: In the Statistical analysis the Authors did not describe how the normality of the data was checked.

Response 4: All the data were normally distributed as verified by Levene’s test. The test has been inserted into the text

Point 5: Why did the Authors the shoot multiplication and root induction present from one representative trial (lines 186-187) but not as the average of the three trials.

Response 5: The average of the three trials was only necessary to establish the survival rate of acclimatized plants because at least three data per treatment are required for statistical comparisons. for the other parameters related to shoot multiplication and root induction we had a good amount of data available that does not require mixing the three experiments

Point 6: Lines 195-198: "Nevertheless, plants in TIS showed the lowest number of shoots per cluster and, some hyperhydricity symptoms were observed, especially at the base of the clusters (Fig. 2). For these last two reasons, the micropropagation in jar system was chosen to produce M. maritima shoots to be used for the subsequent experiments of rooting induction." What was the percentage of hyperhydricity shoots? The hyperhydricity shoots are not visible on the Figure 2.

In my opinion, the shoots micropropagated in temporary immersion system (Plantform) were better quality and should be used also for the rooting. Moreover, the above sentences (lines 195-198) contradict the results presented in the Discussion (lines 329-331: "The liquid culture in temporary immersion system (TIS) improved the dimension of micropropagated shoots, increased the biomass (both fresh and dry), and allowed to obtain longer leaves with longer and wider leaf blades, in comparison with the common jar system with agarized semisolid substrate."

Response 6: The percentage of shoot hyperhydricity (17 ± 2%) was included in the text. The sentence about the use of shoots for rooting trials has been expanded and modified to clarify the reasons for the choice.

Point 7: Line 327-328: "The shoot number per cluster observed in semisolid culture was comparable to that registered to Park et al. [5],...." but Park et al. [5] and Song et al. [6] obtained a much higher multiplication rate after using nodes as explants. Therefore, the use of the temporary immersion system for the multiplication of Mertensia maritima in present study did not improve the micropropagation of these species but it affect the shoot rooting (although, the results also similar to those obtained by Park et al. and Song et al.) and plant acclimatization.

Response 7: Exactly: the use of the temporary immersion system for the multiplication of Mertensia maritima in present study did not improve the micropropagation of these species but the rooting in Plantform affects the shoot rooting and plant acclimatization.

Point 8: The survival rate of the shoots after acclimatization in green house was very low (3-5%) and extremely unsatisfactory, therefore, Mertensia maritima plants can not be obtained on an industrial scale.

Response 8: the best survival rate of the shoot after acclimatization were obtained in plant rooting in Plantform and cultured in growth chamber (56.7 %). After six weeks of acclimatization, the plants could be transferred in greenhouse or outdoors, so it is possible to organize a production of M.maritima plants on an industrial scale.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I still have a few queries in response to the Authors.          

Point 1. Firsty, I have a question regarding to the selection of an explants. Why did the Authors choose microcuttings for the study?

The Authors responded: "the starting material were mother plants grown in pots. In our experience, the surface of 1 cm long cuttings (microtalee) is easily sterilized."

My comment: My question was not about the starting material but about the explants used for the micropropagation. Park et al. [5] and Song et al. [6] obtained a much higher multiplication rate after using nodes as explants. Why did not the Authors choose these explants?

Point 2. Semi-solid medium with 0.75% agar - in my opinion this medium was solid but not semi-solid medium.

The Authors responded: "the substrate is gelatinous and therefore semi-solid."

My comment: What is the difference between solid and semi-solid media for the Authors?

Based on physical form, media are classified as solid, semi-solid, and liquid. The key different between solid media and semi-solid media is that solid media contain a higher concentration of the agar (about 0.7%) while semi-solid media contain a lower concentration of agar.

Point 3. In the Statistical analysis the Authors did not describe how the normality of the data was checked.

The Authors responded: " All the data were normally distributed as verified by Levene’s test. The test has been inserted into the text."

My comment: Levene’s test is used to analyze for equality of variance. What test was used to assess the normal distribution of the variables?

Point 4. Why did the Authors the shoot multiplication and root induction present from one representative trial (lines 186-187) but not as the average of the three trials.

The Authors responded: " the average of the three trials was only necessary to establish the survival rate of acclimatized plants because at least three data per treatment are required for statistical comparisons. for the other parameters related to shoot multiplication and root induction we had a good amount of data available that does not require mixing the three experiments."

My comment: I don't agree with the Authors. In all studies are necessary at least three repetitions per treatment for statistical comparison.

Point 5. Line 327-328: "The shoot number per cluster observed in semisolid culture was comparable to that registered to Park et al. [5],...." but Park et al. [5] and Song et al. [6] obtained a much higher multiplication rate after using nodes as explants. Therefore, the use of the temporary immersion system for the multiplication of Mertensia maritima in present study did not improve the micropropagation of these species but it affect the shoot rooting (although, the results also similar to those obtained by Park et al. and Song et al.) and plant acclimatization.

The Authors responded: "Exactly: the use of the temporary immersion system for the multiplication of Mertensia maritima in present study did not improve the micropropagation of these species but the rooting in Plantform affects the shoot rooting and plant acclimatization."

My comment: In this study the temporary immersion system slightly improve number of roots per shoots by only compared to the result obtained by Song at al. (14 vs. 11.3). In study of Park et al. this parameter was 25.4. Additionally, the roots length in the present study was much less (9 mm, Table 2) than that obtained by Park et al. and Song et al. (4.2 cm and 5.1 cm, respectively).

Point 6. The survival rate of the shoots after acclimatization in green house was very low (3-5%) and extremely unsatisfactory, therefore, Mertensia maritima plants can not be obtained on an industrial scale.

The Authors responded: "the best survival rate of the shoot after acclimatization were obtained in plant rooting in Plantform and cultured in growth chamber (56.7 %). After six weeks of acclimatization, the plants could be transferred in greenhouse or outdoors, so it is possible to organize a production of M.maritima plants on an industrial scale."

My comment: The survival rate of 56.7% when the plantlets were cultured in growth chamber. What was the survival rate after transferring the plantlets to greenhouse or outdoors?

Author Response

Point 1: My comment: My question was not about the starting material but about the explants used for the micropropagation. Park et al. [5] and Song et al. [6] obtained a much higher multiplication rate after using nodes as explants. Why did not the Authors choose these explants?

The Authors responded: “The Mertensia clusters consisted of shoots which were 1.0 – 1.5 cm long with 3 – 4 leaves and nodes. The microcuttings used in our experiments had nodes: one node for each leaf. This material was very homogeneous, abundant and optimal for experimental tests.

Point 2: 

My comment: What is the difference between solid and semi-solid media for the Authors?

Based on physical form, media are classified as solid, semi-solid, and liquid. The key different between solid media and semi-solid media is that solid media contain a higher concentration of the agar (about 0.7%) while semi-solid media contain a lower concentration of agar.

The Authors responded: “semisolid was replaced with solid”

Point 3: My comment: Levene’s test is used to analyze for equality of variance. What test was used to assess the normal distribution of the variables?

The Authors responded: “Shapiro-Wilk test. The text was been added”

Point 4: My comment: I don't agree with the Authors. In all studies are necessary at least three repetitions per treatment for statistical comparison.

The Authors responded: “the text has been changed to clarify statistical analysis: Data were statistically analyzed by one-way ANOVA followed by Fisher’s probable least-squares difference test with cut-off significance at p ≤ 0.05. All the data were normally distributed as verified by Shapiro-Wiks and Levene’s test. The shoot multiplication and root induction trails were repeated in triplicate. In each trial, forty explants per treatment were analyzed. The data about the survival rate of acclimatized plants were reported as the average of three trials performed.”

Point 5: In this study the temporary immersion system slightly improve number of roots per shoots by only compared to the result obtained by Song at al. (14 vs. 11.3). In study of Park et al. this parameter was 25.4. Additionally, the roots length in the present study was much less (9 mm, Table 2) than that obtained by Park et al. and Song et al. (4.2 cm and 5.1 cm, respectively).

The Authors responded: “we do not claim that the rooting in Plantform is better than that observed by Park et al., but that in our experiment, the rooting in Plantform is better than the rooting in the jar. (see Line 344-346: The concentrations of 4 µM IBA and ½ MS salts were chosen for in vitro rooting because they were already used successfully to induce the formation of adventitious roots in M. maritima, as showed in previous studies [5, 6]. In our study, the rooting occurred both in solid and liquid substrate, but in TIS culture the roots were more abundant and longer.)

Point 6: The survival rate of 56.7% when the plantlets were cultured in growth chamber. What was the survival rate after transferring the plantlets to greenhouse or outdoors?

The Authors responded: “After acclimatization in the climatic chamber the plants were transferred to the open air and their survival rate was 100%”

Author Response File: Author Response.docx