The Effect of Harvest Date on Temporal Cannabinoid and Biomass Production in the Floral Hemp (Cannabis sativa L.) Cultivars BaOx and Cherry Wine

Round 1

Reviewer 1 Report

The present paper provides a straightforward model for the temporal accumulation of CBD and THC as well as establishing harvest timing recommendations. There are some interesting and useful results in this paper, but the manuscript needs some work before publication. 

 

I have a few specific observations.

 

Introduction needs more approach on the problematic. Although it is an interesting data, personal communication is unacceptable. 

 

Results should be presented in the order they appear in Materials and Methods.

 

Massuela et al. found that the optimum time of harvest was around nine weeks of flowering. They did not observed a yield plateau, but it was a different methodology applied and they observed inner plant variability indicated significantly higher total CBD concentrations in the top fraction and significantly lower total CBD yields in the low fraction of the plant.

Here the authors did not separate leaf from floral material as farmers in the region thresh plants and sell both flower and leaf combined. Why this information only appears in the conclusion?

 

The authors state:

“the floral and leaf material was stripped from the stalk by hand and the stalk was discarded. The weight of the floral and leaf material was recorded and a representative sample was submitted for cannabinoid analysis… …The protocol for the analysis went as follows: 0.1 grams of dried ground plant material…” How representative was this 0.1 grams from flower and leaf tissue? Doesn’t it influence the analysis?

 

 

Table 2 and Table 4 are lacking the legend identification.

 

Finally, the English usage needs work  The syntax is mostly correct, but there are places where the word usage is either misleading or confusing.  This could be easily remedied by an edit pass.

 

Overall, I think the work presented has potential to be a useful contribution, but there is need for a more rigorous presentation of details underlying strategy and more effort going into prioritizing what comparisons and details are being documented.

Author Response

 

Introduction needs more approach on the problematic. Although it is an interesting data, personal communication is unacceptable. 

 We understand that having a published reference for the problem is ideal; however, the NCDA never published this data. Furthermore, North Carolina has since moved under the umbrella of the USDA’s hemp program – the NCDA no longer manages licenses or production and has since dissolved the NCDA Hemp Program. Consequently, even if they wanted to publish these data, they no longer have the staff/program to do so.

 

Results should be presented in the order they appear in Materials and Methods.

Order of data collection as described in Materials and Methods: Description of biomass processing and data collection (Line 129 – 130); Description of Cannabinoid analysis (Line 130 – 153)

 

Results are presented as: biomass production (Table 2, Figure 1); Total THC (Table 4, Figure 2); Total CBD (Table 5, Figure 3), Relationship between CBD and THC (Figure 4, Table 6).

 

Massuela et al. found that the optimum time of harvest was around nine weeks of flowering. They did not observed a yield plateau, but it was a different methodology applied and they observed inner plant variability indicated significantly higher total CBD concentrations in the top fraction and significantly lower total CBD yields in the low fraction of the plant.

Here the authors did not separate leaf from floral material as farmers in the region thresh plants and sell both flower and leaf combined. Why this information only appears in the conclusion?

 

Comparing results between indoor production systems (Massuela et al.) and outdoor production systems (paper in review) are not appropriate given the differences in systems. It is akin to comparing greenhouse heirloom tomato production (high value, low production) to field grown paste tomato systems. Furthermore, Massuela et al. used artificial light manipulation with set photoperiods, which can certainly alter biomass production/accumulation. Finally, these authors used a different cultivar. As observed in the paper under review, differences in biomass accumulation (here we observed a difference in the rate of biomass accumulation) exist between cultivars. Consequently, to compare results from a different cultivar, grown in a different production system are not appropriate.

 

We have added a sentence in the introduction (Line 80-81) that specifically defines how farmers in the region process hemp for cbd extraction.

 

The authors state:

“the floral and leaf material was stripped from the stalk by hand and the stalk was discarded. The weight of the floral and leaf material was recorded and a representative sample was submitted for cannabinoid analysis… …The protocol for the analysis went as follows: 0.1 grams of dried ground plant material…” How representative was this 0.1 grams from flower and leaf tissue? Doesn’t it influence the analysis?

 A sentence was added (line 132) detailing that ~50g samples were submitted for cannabinoid analysis. Yes, the 0.1g sample for analysis is low, but that is what is required for HPLC analysis.

 

Table 2 and Table 4 are lacking the legend identification.

Please clarify legend identification. Both tables contain footnotes and title.

 

Finally, the English usage needs work  The syntax is mostly correct, but there are places where the word usage is either misleading or confusing.  This could be easily remedied by an edit pass.

We have gone over the paper again and cleaned up language when deemed confusing/misleading. If there are specific sentences or statements that require further editing please specify.

 

Overall, I think the work presented has potential to be a useful contribution, but there is need for a more rigorous presentation of details underlying strategy and more effort going into prioritizing what comparisons and details are being documented.

 

Reviewer 2 Report

Minor corrections as in the MS

Comments for author File: Comments.pdf

Author Response

We thank the reviewer for the thorough edits pertaining to the manuscript grammar and flow- this will certainly make the ms stronger. We have made appropriate changes. The one change we did not make was calling an effect "insignificant" instead of "not significant". While this may seem minor, there are differences in the terms: not significant (or nonsignificant) implies the source of variation did not have a statistical effect (p>0.05); insignificant generally implies unimportant, without statistical implications.

Reviewer 3 Report

The phenomenon of a decrease in THC levels in the end of harvesting period is well known. That is why Health Canada requires measurements of cannabinoids at 50% seed maturity. Frankly, I do not see any novelty in the manuscript. Two cultivars are not enough to speak about the absence of cultivar specific response.

The main problem of the manuscript is that the authors studied hemp, where THCA synthases are absent/inactive. So, the conclusion about the behavior of THC accumulation is based on the promiscuity (decrease in?) of CBDA synthases. Comparison to high THC cultivars would allow to observe the same effect in behavior of CBD - if this is due to the decrease in promiscuity of the enzymes, then you would observed steady increase in THC in high THC varieties, with CBD dropping in the end of the flowering cycle. As far as I know this does not happen. So, what is the mechanism then? It would be good to demonstrate CBN concentrations, to exclude higher rate of conversion from THC in response to senescence.

Another problem with the use of hemp cultivars is the low level of THC - thus the chance of mistake is high. I did not find anywhere what number of samples were done per data point? It says that plots had 20 plants per plot - was this the number of independent samples as well?

Author Response

Response to Reviewer 3:

The phenomenon of a decrease in THC levels in the end of harvesting period is well known. That is why Health Canada requires measurements of cannabinoids at 50% seed maturity. Frankly, I do not see any novelty in the manuscript. Two cultivars are not enough to speak about the absence of cultivar specific response.

We understand that this information may be well known for Canadian producers growing grain hemp, this information was not well known for those farmers producing floral hemp for cannabinoid extraction. Note that this research was conducted to address a specific issue with North Carolina farmers not understanding the appropriate time to harvest their crop (Line 76 defines problem of non-compliant samples in the state).

 

The main problem of the manuscript is that the authors studied hemp, where THCA synthases are absent/inactive. So, the conclusion about the behavior of THC accumulation is based on the promiscuity (decrease in?) of CBDA synthases. Comparison to high THC cultivars would allow to observe the same effect in behavior of CBD - if this is due to the decrease in promiscuity of the enzymes, then you would observed steady increase in THC in high THC varieties, with CBD dropping in the end of the flowering cycle. As far as I know this does not happen. So, what is the mechanism then? It would be good to demonstrate CBN concentrations, to exclude higher rate of conversion from THC in response to senescence.

Please refer to the objectives of this study, which were to model temporal accumulation of CBD and THC in field-grown floral hemp in North Carolina and establish harvest timing recommendations to minimize non-compliant crop production. High THC cultivars are illegal to produce and research in our state and federally illegal, furthermore it is outside of the scope of this research as defined by the specific objectives and production system.

Another problem with the use of hemp cultivars is the low level of THC - thus the chance of mistake is high. I did not find anywhere what number of samples were done per data point? It says that plots had 20 plants per plot - was this the number of independent samples as well?

Thank you for catching this. We added line 132-134:  “A total of 48 samples per cultivar × harvest date (3 plant subsamples × 4 replicates× 2 locations × 2 years) were used to quantify cannabinoid content.”

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

n/a

Author Response

Tables corrected.

Author Response File: Author Response.docx