Plant Material and Growing Conditions
Three medicinal cannabis genotypes provided by Cann Group Ltd (https://www.canngrouplimited.com/, accessed on 22/03/2024) were employed in this study. These genotypes included one high CBDA strain, "Cannatonic," and two high THCA strains, "Hindu Kush" and "Northern Lights" (previously referred to as "CBD1," "THC6," and "THC1," respectively) [37].
All plants were cultivated in a secure facility approved by the Australian Government Department of Health and Aged Care Office of Drug Control (ODC). The experiments were carried out under a Commonwealth license and associated permits. Temperature and humidity were maintained at 25°C and 50%, respectively. The plants were grown in controlled environments (CE) throughout their life cycle, and they were moved within the CEs on a weekly basis during the flowering period to account for any environmental variations.
The cloning and propagation method used has been previously documented [37]. Experimental plants were cloned from donor mothers. Approximately 15 cm of new growth stems were excised from the mother plants. All leaves on the stem sides were removed, leaving only the top leaf bunch. The bottom of the stem was cut diagonally across a node, creating a clone approximately 12 cm in height. The top leaf bunch was trimmed to the height of the smallest emerging leaf to reduce water loss and prevent overlapping in the propagation dome. The bottom 1 cm of the stem, where roots would form, was lightly scraped with a scalpel, dipped in hormone gel (Clonex Purple, Yates, DuluxGroup, Clayton, Australia), and placed in an organic propagation cube (Eazyplug CT12, Goirle, The Netherlands, eazyplug.nl).
Once the propagation tray was filled with new clones, it was positioned in a propagation dome (Smart Garden heavy-duty 3-piece propagation kit, Epping Hydroponics) for 14 days. The clones were subjected to an 18-hour light/6-hour dark (18L:6D) photoperiod in a growth cabinet (Conviron A2000, Conviron Asia Pacific Pty Ltd, Grovedale, Australia) at a light intensity of 100 µmol m² s⁻¹ and a temperature of 25°C. Humidity was gradually reduced over the 14-day propagation period. Plants with established roots were then transplanted into 1.8-litre pots containing a 30:70% blend of perlite and coco-coir with an electrical conductivity (EC) of <0.5 mS/cm (Professors Nutrients, Australia). Subsequently, the plants were transferred to controlled flowering environments under a photosynthetically active radiation (PAR) of 600 µmol m² s⁻¹ (Heliospectre Grow Light, LX602C) and maintained under an 18L:6D photoperiod for an additional 5 days as a hardening period to acclimate to the new environment. After this period, the photoperiod was switched to one of the 6 treatments described below and flowering was initiated. The temperature was kept at 25°C, and blackout curtains prevented light leakage. The plants were watered and fed using a commercial fertigation recipe with an electrical conductivity (EC) of 2.2 mS/cm and a pH of 6.
Treatments
Each treatment contained 7 replicates of each of the 3 genotypes in a fully randomised design. The six zones were programmed to one of each of the following photoperiods with different timing applications of Far-Red as presented in Table 3. All plants were maintained in these treatments for 40 days, which was the day after cloning (DAC) 70, which is the end point of the flowering treatments.
The six programmed zones were controlled using Heliospectre Software (heliospectra.com). A description of each six light spectrum treatments is described in Table 4. The light intensity drops for all treatments from hour 8 because the maximum power (uMol m2 Sec-1) of the red light band (660nm) is more powerful than the maximum of the FR band (735nm) therefore, in order to achieve a low ratio, the FR had to be turned up and the red band down. Treatments not receiving FR in the light period were treated the same to ensure that the daily PAR was consistent between treatments.
Table 3. Six treatments applied. Seven replicates per genotype were included for each treatment.
Treatment 1 (Light/Dark)
|
Total FR
hours
|
Name
|
Description
|
10L:14D
|
0
|
10L
|
Stayed under 10L:14D for the duration of the experiment
|
10L:14D (FR 8-10)
|
2
|
10L_2
|
Applied FR light at photoperiod hours 8-10
|
10L:14D (FR 10-12)
|
2
|
10L_2D
|
Applied FR light at photoperiod hours 10-12 (in the dark)
|
10L;14D (FR 8-12)
|
4
|
10L_2_2D
|
Applied FR light at photoperiod hours 8-12 (2h in the light 2h in dark)
|
12L:12D
|
0
|
12L
|
Stayed under 12L:12D for the duration of the experiment
|
12L:12D (FR 10-14)
|
4
|
12L_2_2D
|
Applied FR light at photoperiod hours 10-14
|
Table 4. The PAR, % of each colour in the total spectrum and ratios for the treatments
|
|
|
%
|
|
Treatment
|
PAR
|
Hour
|
B
|
R
|
FR
|
W
|
R/FR
|
|
790
|
0-8
|
9.25
|
55.88
|
0.07
|
34.81
|
833.63
|
10L
|
425
|
8-10
|
19.71
|
5.95
|
0.14
|
74.19
|
41.68
|
|
0
|
10-12
|
0.00
|
0.00
|
0.00
|
0.00
|
-
|
|
790
|
0-8
|
9.25
|
55.88
|
0.07
|
34.81
|
833.63
|
10L + 2L
|
425
|
8-10
|
17.27
|
5.22
|
12.52
|
65.00
|
0.42
|
|
0
|
10-12
|
0.00
|
0.00
|
0.00
|
0.00
|
-
|
|
790
|
0-8
|
9.25
|
55.88
|
0.07
|
34.81
|
833.63
|
10L + 2D
|
425
|
8-10
|
19.71
|
5.95
|
0.14
|
74.19
|
41.68
|
|
0
|
10-12
|
0.00
|
0.00
|
100.00
|
0.00
|
-
|
|
790
|
0-8
|
9.25
|
55.88
|
0.07
|
34.81
|
833.63
|
10L + 4
|
425
|
8-10
|
17.27
|
5.22
|
12.52
|
65.00
|
0.42
|
|
0
|
10-12
|
0.00
|
0.00
|
100.00
|
0.00
|
-
|
|
790
|
0-10
|
9.25
|
55.88
|
0.07
|
34.81
|
833.63
|
12L
|
425
|
8-10
|
19.71
|
5.95
|
0.14
|
74.19
|
41.68
|
|
425
|
10-12
|
19.71
|
5.95
|
0.14
|
74.19
|
41.68
|
|
790
|
0-8
|
9.25
|
55.88
|
0.07
|
34.81
|
833.63
|
12L + 4
|
425
|
8-10
|
19.71
|
5.95
|
0.14
|
74.19
|
41.68
|
|
425
|
10-12
|
17.27
|
5.22
|
12.52
|
65.00
|
0.42
|
|
0
|
12-14
|
0.00
|
0.00
|
100.00
|
0.00
|
-
|
Measurements
All Red to Far Red (R:FR) ratios were determined using a LightScout Red/Far Red Meter (spec-meters.com)/ (accessed on October 19, 2023).
Flowering development was assessed on a weekly basis starting from Day After Cloning 37 (DAC 37) to ascertain the presence or absence of pistils (scored as 1/0) and trichomes (scored as 1/0). Plant height was measured weekly, commencing from DAC 30.
Harvesting: The harvest took place on DAC 70. Plants were excised at the base, and then the whole plant was weighed (whole plant FW). The large fan leaves were removed, and the flowers were manually stripped from the stem and trimmed using a mechanical trimmer (TrimPro ROTOR, Canada). The trimmed flowers were re-weighed (trimmed flower fresh weight) and placed into a foil tray. The flowers were dried in a dedicated drying room at 21 °C and 50% humidity until no further reduction in weight was observed (9 days). The samples were then re-weighed, and the total flower dry weight (g plant−1) was calculated.
Analytics
The methodology for quantifying cannabinoids has been previously documented [25]. For each of the six treatments and control groups, four biological replicates were analyzed for THCA and THC (in the cases of Hindu Kush and Northern Lights) and CBDA and CBD (for Cannatonic), encompassing three different genotypes. Total THC and CBD was then calculated with the following formulae: Total THC = THC + (THCA*0.877) and Total CBD = CBD + (CBDA*0.877).
From each individual plant, three florets were randomly selected from the dried subsample of flower material and finely ground into a powder using liquid nitrogen. A 0.1-gram sub-sample was employed for cannabinoid quantification, which involved sonication in 100% ethanol using a SONICLEAN instrument (Soniclean®, Dudley Park, Australia) operating at 50/60 Hz for 30 minutes, followed by centrifugation at 10,000 rpm for 10 minutes. The resulting ethanolic extracts were preserved at -10 °C until needed. Subsequently, these extracts were diluted and subjected to analysis through high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QToFMS, Agilent Technologies, Santa Clara, CA, USA). Separation was achieved using a reversed-phase column (Agilent Infinity Poroshell 120, HPH-C18, 2.1 × 150 mm, 2.7 µm, narrow bore LC column, Agilent Technologies, Santa Clara, CA, USA) with mobile phases consisting of methanol-water-acetonitrile and acetonitrile, both containing 0.1% formic acid (v/v). The analysis was executed using Quant Analysis Software 10.2 (Agilent Technologies, Santa Clara, CA, USA), and cannabinoid peaks were identified based on their mass-to-charge (m/z) values and retention times by calibration against cannabinoid standards (Novachem, VIC, Australia).
The total yield of each cannabinoid in g per plant (g plant−1) was calculated as follows:
((%Cannabinoid/100) x Total flower dry weight in grams) = g cannabinoid plant−1
Power Use Calculations
The kWh used by each of the treatments was calculated by an amperemeter. The carbon emissions per kWh for NSW Australia were sourced from The Australian Greenhouse Accounts Factors 2022 [27].
Statistical Analyses
All graphics and statistical analyses were performed in R 3.1 [38]. One way ANOVA’s were performed per variety and Tukey HSD tests were used to identify pair-wise differences between treatments significance for all tests was set at ≤0.05. In circumstances were the ANOVA returned a significant statistic but the Tukey HSD test could not identify difference at the ≤0.05, a Least Significant Differences (LSD) test was performed using the agricolae package in R [38,39].