Establishment of an Efficient In Vitro Propagation Protocol for Cannabis sativa L. subsp. ruderalis Janish
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThese experiments have identified an efficient micropropagation scheme for obtaining plants in vitro and later in vivo, which meets the in vitro adaptation challenges caused by photoperiodic independence and short cycles, and has broad application prospects.
There are a number of issues that must be resolved before publication can be considered. If the following questions are well addressed, this reviewer of cloning reproduction considers the important contribution of this paper to be important for cloning propagation. 1, the summary can be more concise and clear:
1, the summary can be more concise and clear:
2. Chart quality needs to be improved:
3. In the introduction section, the background information is not detailed enough.
4. Limitations of the study can be discussed.
5. Complete analysis of the results and more profound conclusions;
6. The next research direction and application can be described in more detail.
Author Response
Dear reviewer 1, attached are the answers to your comments and suggestions.
Best regards
Claudia Ruta
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
The study is worth to explore new propagation techniques for hemp or other crops those have genetical shortcomings.
I have highlighted some of the points in comments to bring author's attention to use scientific technical language. These are a few examples. Please go thoroughly and make the additional changes in the writing.
Comments for author File: Comments.pdf
Scientific soundness is important for publication. Therefore, please make efforts to improve the scientific writing.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe MS entitled “ Establishment of an efficient in vitro propagation protocol for Cannabis sativa L. subsp. ruderalis Janish” with authors Giuseppe N. Basile, Luigi Tedone, Cataldo Pulvento, Giuseppe De Mastro and Claudia Ruta presents new and interesting data but needs major improvement to be published.
Cannabis plants (Cannabis sativa L.) belong to the Cannabidaceae family and are cultivated for fiber, oil and medicinal purposes. Three subspecies are known — Cannabis sativa ssp. sativa (L.), Cannabis sativa ssp. indica (Lam.) and Cannabis sativa ssp. ruderalis (Janish). Cannabis sativa is the most widely distributed variety, growing in both tropical and temperate climates. Cannabis can be propagated in open or closed farming systems using seeds, clonal propagation or tissue culture methods. In vitro clonal propagation is the preferred propagation method. Through in vitro propagation of the mother plant, the desired qualities are preserved. Most in vitro studies have achieved successful callus induction of various cannabis explants, and in vitro direct regeneration from hypocotyls, cotyledons and leaves has also been achieved. Despite these successes, more studies need to be conducted to establish higher indirect regeneration rates. In this regard, the research done in the presented manuscript deserves attention.
The article follows the order required by the journal: Introduction, Materials and Methods, Results, Discussion, Conclusions. Abstract is informative; the Introduction is good and to the point to the purpose; the purpose of the study is clearly stated. Over 70% of the cited literature is from the last 10 years, оf which over 70% are from the last 5 years. It is essential to improve Material and Methods, and data presentation.
I have some comments on the text:
L65 – L72 - In my opinion, this passage can be dropped from here and transferred to another place in the text, for example, Discussion
L155 - Is the indicated BM medium different from the basic MS medium, and if different, please describe the differences
L173 - What information does the parameter „leaves per each shoot” provide, please describe
L221 - How are the female plants determined, please describe.
L223 - In my opinion, this table (Table 1) can be dropped and the result described in the text.
L339 – Figure 4a - I recommend the authors to include a better figure (if possible). GA3 - it is correct to write the 3 in subscript.
L390 – In Material and Methods it is not stated that the authors used a concentration of 20 mg/l MT. If this is a citation, please include the authors.
L479 - I recommend that the author's correct the Conclusion, as it largely repeats the Abstract
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe Authors set themselves an ambitious aim - to develop an efficient method of micropropagation from explants taken from almost mature/postjuvenile hemp 'Finola' plants. However, the manuscrypt contains so many mistakes and discrepances that it is completely unsuitable for publication. I'm afraid I won't point out all mistakes…
Please take into consideration comments and suggestions:
Keywords
The same phrase as in the title (in vitro)! What about micropropagation in the keywords?
Introduction:
Please provide more information. Is ‘Finola’ local variety, botanical variety (var.) or cultivated variety (cv.; cultivar)? What are the traditional methods of propagation of ‘Finola’? Is it propagated by seeds and/or cuttings? Why such methods are unsuitable? Why propagation through certified seeds is not recommended? I think such method is much much cheaper than micropropagation? It is unclear why „above-mentioned characteristics make “Ruderalis” difficult to multiply” Why day-neutral hemps are more difficult/recalcitrant to clone than hemps sensitive to photoperiod (SD)? Please clarify!
v. 32-36 “Ruderalis” strains also have 32 a unique characteristic called "auto-flowering," which means they automatically transition(???) from the vegetative stage to the flowering stage based on their age, rather than relying on light cycles [5]. Instead, the cultivation cycle of hemp is typically annual and photo(?)-dependent(?), strongly linked to seasonal trends and consequently influenced by the number of hours of light and darkness per day. Unclear. If ‘Finola’ is "auto-flowering"/ day-neutral plant than why is influenced by the number of hours of light and darkness per day?
v.39 While auto-flowering strains containing ruderalis genetics(???) are renowned …
Materials and Methods
v.82 Information about ‘Finola’ should be moved to Introduction
v.82 short length (???)
v. 86-90 from certified Finola seeds.
2.2 Explant source
Certified Finola seeds(!) were sowed directly in vitro or in controlled conditions to obtain mother plants. The buds were then excised to initiate micropropagation by axillary shoots.
! – the same information. When were the seeds sown? How old were seedlings/mother plants which were the source of buds/explants?
v. 125- 125 The controlled greenhouse, located at the Department of Soil, Plant and Food Sciences (University? Town? Country?????), had a temperature control system, maintained between 18°C and 126 25°C, 16-hour photoperiod of natural light per day and relative humidity of 85%; on the 127 other hand, the thermostat-controlled cabinet had illumination with white LED lights with a spectrum between 400 nm and 700 nm at an intensity of 80 µE s-1 m-2, photoperiod set at 24 h light and temperature of 22°C ± 1°C.
16-hour natural light per day in Italy!? At this latitude? Why 24h/0h photoperiod was set?
v.111 and a light intensity of 50 μE s-1 m-2. The photoperiod was set at 16 h of light and the temperature at 22°C ±1°C.
What was the length of dark/night? 8h? Then it was 16 h/8 h (d/n) photoperiod, not 16 h photoperiod!
The einstein (E) is an obsolete and not SI unit. Use SI unit i.e. µmol m-2 s-1". Was it data for PhAR? Intensity of light - the name for irradiance is measured in (W/m2) not in µE!
v.140. adding sucrose (20 gr L-1) and growth phytoregulators of the cytokinin group to stimulate the multiplication. The cytokinins used were Metatopolin (MT) [6-(3-hydroxybenzylaminopurine) in the concentration of 0.5 mg L-1 or 6-Benzylaminopurine (BAP), employing a concentration of 0.05 mg L-1.
Why those cytokinins and concentrations were chosen? gr?
v. 148 with sodium hypochlorite (NaClO 14%) at 1.4% for 20 min Unclear - 1.4% or 14% solution?
v.139 BM culture medium, solidified with agar (7 g L-1) What was the composition of BM?
v. 162-Benzylaminopurine (BAP) (0.05 mg L-1);
-Metatopoline (MT) (0.5 mg L-1); 163
-Thidiazuron (TDZ) (0.4mg L-1) + 1 naphthalenacetic acid (NAA) (0.2 mg l-1); 164
-Thidiazuron (TDZ) (0.4 mg L-1) + 2,3,5- Triiodobenzoic acid (TIBA 1 g L-1).
Why those PGRs and concentrations were chosen? Were the media autoclaved? When were PGRs added, after or before autoclaving?
v.176 and some other verses with a photoperiod of 16 h light. 16 h/8 h (d/n)
v. 180 Around 200 seedlings, which had reached a minimum length of 3 cm, were transplanted seedlings? Not shoots or microshoots?
v.189- 192 rate and the length and number of roots were evaluated.
The pH of all the media was adjusted to 5.6–5.8 with 1M KOH and then at 121 °C for 20 min. - Why is it written here and not next to the information about the composition of the food? chaos!
The culture of the explants(??) was instead(???) carried out… expl were prepared and placed onto... under a horizontal laminar flow hood to ensure the necessary sterile conditions. this seems obvious?
v. 196 What the radical apparatus is?
v 197 pots (8 cm2)???
v. 215 3.1 In vitro germinated seeds Germination of seeds in vitro?
v.222 showed a good development(?) in length and number of nodes to induce(?) the multiplication nieszczęśliwe statement; length of node or of shoot?
Table 1 is unnecessary. So little information can be included in the text, especially since one (42%) was already mentioned
Figure 1. Cotyledon emission(???) and appearance of true leaves small photo, not clear :/
v. 250-1 The growth and multiplication responses of the shoots of Finola, expressed as mean length, number of nodes and mean multiplication index (M.M.I.) in the presence of the two different phytoregulators tested are shown in Table 2.
You should make a statement and not refer the reader to the table to do it themselves. The same information in the text and table title!
v.278-280 In Table 3, the values of the different growth parameters measured under the two different conditions of mother plant development at the time of the first female flower appearance 279 were shown. In addition, the number of days from sowing to flowering and the percentage of male and female flowers were recorded. Unnecessary introduction - the same information in the text and table title!
Table 3 development (of what?) Flower apparence?? (dd) What kind of flowers? What is dd unit?
Length of what??? (cm) Stem diameter 45mm=4,5cm!?!?!?!?!?!?!?!?!?!?!?!
Mean of three subcultures ??? Were 3 subcultures carried out in different time?
Figure 3 Add a bar [cm] in each photo, How old were plants at the time of the photo was made?
v. 322 The length was more than twice (47.1 cm vs 15.9) and the stem diameter was almost twice (45 mm compared to 24 mm) 4,5cm v 2,4cm???? The same data in table and text! So why did you create the table??
In Table 4, the effects of nutrient media and different phytoregulators tested on the growth, mean multiplication index, and hyperhidricity of Finola shoots, obtained from buds of plants grown in the growth cabinet, are shown. Unnecessary introduction - the same information in the text and table title and captions!
v. 342 there weren’t significant difference among all the parameters measured (???)
You didn't measure differences among parameters but in terms of the measured parameters; significant differences between media influence on measured traits were not proven
The same mistake as earlier - the same data in table and text! So why did you create the table??
White/Pink - why such type of light was applied/chosen? Clarify in M&M
Fig 4b, Add a bar [cm]. What do the numbers 1-4 under the explants mean?
Discussion
v. 372 The research carried out aimed to overcome several limits linked to the vegetative propagation of C. sativa L. subsp. ruderalis Janish. var. Finola. I still haven't found out what the limits are?
v. 373 This variety is dioecious, meaning it has separate male and female plants and is Hmm, are there monoecious hemps?
v. 379 The comparison of the sterilization test conducted on a sample of 100 seeds between two sterilizing agents the same information in M&M
v. 386 HgC2.???
v. 389 metatopolin (MT) 20???? mg L-1
Based on the results overview shown - overloaded style :/
v. 429-30 The results of the current study showed an increase of the length of the shoots (2.8 cm), in comparison with the combination of BAP+ GA3 or MT + GA3 (2.0 cm and 1.5 cm respectively) The same data in Discussion, results, Table!!! Rephrase the statement
Conclusions
v. 485-6 MB x(?) TDZ + NAA MS medium supplemented with TDZ (?) and NAA(?) ?
using a low concentration(?) of IBA under red and blue light Specific parameters should be provided (concentration, PPFD, etc.)
Summarizing, the aim of the study was ambitious and and some quite interesting results were obtained. However, the manuscript has many mistakes and disadvantages which preclude its acceptance. I'm not an English specialist but I think the manuscript is written in awkward English :/ Perhaps this is the Authors' first work? Before submitting a new version of the manuscript they should follow other published works on micropropagation. They should also ask for help a person more experienced in writing the scientific texts and someone with fluent English.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsТhanks to the authors for considering my comments
Author Response
Thanks to you for the useful suggestions
Claudia Ruta
Reviewer 4 Report
Comments and Suggestions for AuthorsThe Authors greatly improved their manuscript and I accept it.
I suggest the only one, small change: 15/9 h (d/n) into 15h/9h (d/n) , 16/8 h to 16h/8 h, etc.
Best wishes :)
Author Response
Thank you for the suggestion.
The small change has been made.
Regards
Claudia Ruta
