Industrial Crops & Products 167 (2021) 113528

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Industrial Crops & Products

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Plant architecture manipulation increases cannabinoid standardization in

‘drug-type’ medical cannabis
Nadav Danziger a, b, Nirit Bernstein a, *
a
Institute of Soil Water and Environmental Sciences, Volcani Center, 68 HaMaccabim Road, P.O.B 15159, Rishon LeZion, 7505101, Israel
b
The Robert H. Smith Faculty of Agriculture, the Hebrew University of Jerusalem, Israel

A R T I C L E I N F O A B S T R A C T

Keywords: A major challenge in utilizing cannabis (Cannabis sativa L.) for modern medicine is the lack of standardization
Cannabissativa throughout the plant of cannabinoids, the unique therapeutic secondary metabolites in cannabis. The present
Cannabinoids study focused on the interplay between plant architecture modulation and standardization of the secondary
CBD
metabolite profile in medical cannabis plants. Secondary metabolism is considerably affected by endogenous and
Defoliation
Pruning
exogenous factors, including positional-developmental aspects and microclimate. We hypothesized that
THC manipulation of the plant architecture, which alter morphological and reproductive development, as well as
micro-climate in the shoot will impact secondary metabolism and spatial standardization of the cannabinoids.
Eight plant architecture-modulation treatments were evaluated for effects on in-planta concentrations and
standardization of the cannabinoids, and on the morpho-physiological state of the plants. Two medical-cannabis
genotypes of ’drug-type’ medical cannabis were analyzed to evaluate genotypic sensitivity. The results reveal
that plant architecture modulation can increase standardization of the cannabinoid profile in cannabis, thereby
supporting the hypothesis. The architectural modulating treatments increased uniformity of cannabinoid con­
centrations in the plant by increasing concentrations at the lower parts of the plant. The cannabinoid profile was
most affected by treatments that had the largest impact on plant structure such as the removal of primary and
secondary branches. Decarboxylation of the cannabinoids in-planta was not affected by structural modulation.
The spatial uniformity of cannabinoid concentrations throughout the cannabis plant is cannabinoid and genotype
specific, and the effect of architecture modulation on cannabinoid standardization is genotype specific.

1. Introduction
Cannabis sativa L. is used by mankind since antiquity as a medicinal
and industrial plant (Zuardi, 2006). Recreational use as a hallucinogenic
drug has led to an almost worldwide ban on research, commercializa­
tion, and utilization for both medical and recreational uses. With resent
changes in regulation, the medical potential of cannabis is increasingly
studied and was reported effective for a wide range of medical condi­
tions (Mouhamed et al., 2018). The extensive therapeutic activity
sources from the diverse chemical profile, which is comprised of hun­
dreds of biologically-active secondary metabolites, including cannabi­
noids, terpenes and flavonoids (Andre et al., 2016). The vast assortment
of active compounds, also bears difficulty, as with each variation in
concentration, composition, or ratios between the metabolites, the
biological activity and its effectiveness may be altered (Russo, 2019).
The medical product commonly administered to patients is the plant
material itself, i.e., intact inflorescences, and therefore uniformity of the
therapeutic-compounds profile between inflorescences in a single plant
and between plants, is needed to ensure a consistent treatment. Since
in-planta variability is not steady between growing cycles, increased
standardization in the plant is required also for maintaining consistency
of therapeutic activity between batches of plant extracts produced by the
pharmacological industry. Utilization of cannabis for modern medicine
therefore involves the challenge of standardization of the secondary
metabolite profile in the plant.
The chemical profile of the cannabis plant is known to be affected by
the genetic background of the plants (Aizpurua-Olaizola et al., 2016;
Janatová et al., 2018), environmental factors (Danziger and Bernstein,
2021; Lalge et al., 2017; Saloner et al., 2021) and morpho-spatial po­
sition in the plant (Bernstein et al., 2019b, 2019a). The cannabidiome is
therefore not uniform within and between plants. The challenge to in­
crease chemical consistency of the cannabis product should therefore

* Corresponding author.
E-mail address: Nirit@agri.gov.il (N. Bernstein).

https://doi.org/10.1016/j.indcrop.2021.113528
Received 21 December 2020; Received in revised form 6 April 2021; Accepted 7 April 2021
Available online 16 April 2021
0926-6690/© 2021 Elsevier B.V. All rights reserved.
N. Danziger and N. Bernstein
tackle each of those parameters. We suggest that spatial-position effects
could be targeted by controlling plant architecture, for production of
inflorescences of similar morpho-spatial characteristics. It should be
considered that positional and environmental effects are intertwined,
because variations in micro-climate between locations in the shoot may
elicit variability in secondary metabolism.
The plant’s environment is composed of many biotic and abiotic
variables that are known to influence plant development and meta­
bolism (Gorelick and Bernstein, 2014; Shiponi and Bernstein, 2021;
Summy et al., 2020; Suzuki et al., 2014). Environmental conditions such
as temperature, light intensity and humidity may impact plants on a
field or a plant level, or be expressed locally on a micro-climate level and
affect plant organs at discrete locations. In modern agriculture, the plant
micro-environment is often changed to achieve a better plant function.
One common technique is pruning, directed at altering plant
morphology and with it microclimate conditions such as better light
exposure and better air circulation and thereby also humidity (Wen
et al., 2019). Different studies reported effects of plant architecture
manipulation (pruning) on microclimate in the plant (Ambroszczyk
et al., 2008) and on yield quantity and quality for different crops
(Alsadon et al., 2013; Ferreira et al., 2018; da Silva et al., 2019) with the
results varying between species and cultivars. In addition to affecting
microclimate of the shoot, pruning alters also the physiological state of
the plant tissues and metabolism, as there are changes in sink-to-source
ratios as well as hormonal changes, for example such that are induced by
the removal of apical dominance (Gautier et al., 2001)
Thus, we hypothesized that reproductive development and meta­
bolism in cannabis will be altered by plant architecture modulation
treatments. If the new generated plant architecture will decrease the
variability of the microclimate in and around the plant, it is possible that
the secondary metabolites profile throughout the plant will be more
uniform. In cannabis, a single study evaluated the effect of architecture
manipulation on plant response in a fiber-type hemp cultivar, and
identified increased seed yield after pruning, effects on the chemical
profile were not assessed (Kocjan Ačko et al., 2019). No information is
available about effects of architectural manipulation on the chemical
profile in ’drug-type’ (medical) cannabis.
Cannabis development is monopodial, with a constant phytomer
structure (Spitzer-Rimon et al., 2019) which is regulated by apical
dominance. This configuration enables the division of the plant’s body
to main stem, ’primary branches’ that are derived from the main stem,
’secondary branches’ that are derived from the primary branches, etc.
While flowering, each meristem develops an inflorescence that could
also be categorized as ’primary’ at the end of the main stem; ’secondary’
at the end of primary branches and so on. By this system a small to
medium plant will produce inflorescences of up to third or fourth order.
Inflorescences also develop along stems and branches from axillary
buds. Plant architecture, and the degree of branching, therefore, have a
direct impact on reproductive development schemes. As of today,
medical cannabis growers utilize several plant architecture techniques
including pruning of the main stem (either once, twice or even three
times), leaf removal (ranging from removal of few old leaves to total
defoliation), branch removals, trellising and any combinations of the
above. While each grower has a different approach for cultivation con­
ditions, it is generally agreed, albeit not supported by empirically tested
evidence, that better light penetration is achieved by defoliation and
branch removal that supports higher yield and chemical standardiza­
tion. Since cannabis is directed to the pharmacological market,
achieving chemical uniformity, and reproducibility, is of outmost
importance and a major challenge.
The present study therefore focused on the interplay between plant
architecture modulation and standardization of the secondary metabo­
lite profile in medical cannabis plants. A goal of the study was to identify
architectural manipulation treatments suitable for minimizing vari­
ability in the cannabinoid profile throughout the plant. We studied
comparatively the response of two cultivars to plant architectural
2
Industrial Crops & Products 167 (2021) 113528
manipulation. The hypothesis guiding the work plan was that plant ar­
chitecture manipulation which alter morphological and reproductive
development, will alter secondary metabolism and its spatial standard­
ization in the plant. To test this hypothesis, we studied how eight plant
architecture modulation treatments involving leaf removal (defoliation)
or pruning (topping or branch removals) affect the cannabinoid profile
and its spatial standardization in the plant, as well as morphological and
physiological characteristics. Besides the contribution to knowledge of
cannabis physiology, the attained information is valuable also for
development of cultivation protocols for increased standardization of
the chemical profile and yield quantity, for the developing medical
cannabis industry.
2. Materials and methods
2.1. Plant material and growing conditions
Two medical cannabis (Cannabis sativa L.) cultivars, ’Himalaya’ and
’Fuji’ (Canndoc LTD, Israel), which are approved for commercial med­
ical use in Israel, were used as a model system of study. Both cultivars
are high THC (tetrahydrocannabinol) (10–16%) and low CBD (canna­
bidiol) (<0.1%) chemotypes with’ Himalaya’ having a longer and
thinner build (Sativa-dominant characteristics) and’ Fuji’ having a
shorter and denser plant structure (Indica-dominant characteristics).
Plants were propagated from cuttings, in coconut fiber plugs (Jiffy in­
ternational AS, Kristiansand, Norway). The rooted cuttings were planted
in 10 L plastic pots in a potting mixture and cultivated in a greenhouse at
the density of 1 plant/m2. Forty uniform plants of each cultivar were
randomly divided to eight groups of five plants and each group was
assigned a different treatment. The plants were cultivated in a certified
commercial cannabis farm in Israel (Canndoc LTD, Israel) in a naturally
lit greenhouse with supplementation of photoperiodic light. At the
vegetative stage, i.e., during the long photoperiod, plants were illumi­
nated 18 h a day (sunlight and then fluorescent lights). For induction of
flowering, the plants were transferred to a short photoperiod. i.e., a
short-day regime of 12/12 h of light/darkness, with black curtains used
to block sunlight when the days grew longer. The plants were trans­
ferred to the short photoperiod following two weeks of vegetative
development in the experimental pots under the long photoperiod.
Fertilizers were supplied by fertigation, i.e., dissolved in the irrigation
solution at each irrigation event (Saloner et al., 2021). Irrigation was
supplied via 1.2 Lh− 1 discharge-regulated drippers (Plastro Gvat, Israel),
two drippers per pot. The volume of irrigation water in each irrigation
event was 500–800 mL/pot/day, set to allow 30% of drainage. The
experiment was terminated 53 (‘Fuji’) and 66 (‘Himalaya’) days
following the transfer of the plants to the short photoperiod. At this time
30% of the trichomes on the flowers were of amber color, which is the
developmental stage accepted for these cultivars as chemical maturity
for medical purposes.
2.2. Experimental treatments
Eight architectural manipulation treatments of various pruning and
defoliation practices commonly used by the growers were evaluated in
the study. The treatments, detailed in Table 1, were initiated 20 days
after the plants were transplanted to the experimental pots and this is
considered day 0 of the experiment.
2.3. Plant morphological development and biomass accumulation
Plant height was measured non-destructively throughout the exper­
iment as the distance between the plant base and the top of the main
stem. In the two pruning treatments, plant height was measured as the
distance between the plant base and the top of the highest branch.
Additionally, at the termination of the experiment, length of selected
branches (i.e., branches 3, 6 and 10, as numbered from the base of the
N. Danziger and N. Bernstein
Table 1
Description of the plant architecture manipulation treatments evaluated in the study.
Plant architecture Description
modulation
treatments
Control No-intervention
Commonly used treatments
Defoliation Removal of 85% of the leaves (top leaves were
kept)
Bottom branches & Removal of all leaves and secondary branches
leaves removal from the bottom 1/3 of the plant
b,c
(BBLR).
BBLR + Defoliation Combination of the 2 treatments above
Branch removal treatments
Primary (1◦ ) Removal of all branches off the main stem
branches removal
Secondary (2◦ ) Removal of all 2◦ branches (including tertiary
branches removal inflorescences) off the 1◦ branches
Pruning treatments (’Topping’)
A single prune Plant decapitation, keeping the 6 bottom
branches
Double prune Plant decapitation, keeping the 6 bottom
branches, followed by a second pruning
(removal of 5 cm from each of the 6 branches)
a
Days are counted from the transition to the experimental setup.
b
BBLR- Bottom branches and leaves removal.
c
Termed ’Lollipopping’ in the cannabis jargon.
plant), was measured as the distance between the node and the tip of the
branch. Stem diameter was measured with a digital caliper (Signet tools
international co., LTD., Shengang Distric, Taiwan), at the middle of the
3rd internode from the plant base. Fresh biomass of fan leaves, stems,
inflorescences and inflorescences leaves for each plant, was measured
destructively at the day of harvest. For evaluation of effects on com­
mercial yield, dry biomass of the inflorescence, following trimming the
tips of the inflorescence leaves as is practiced in the cannabis industry,
was measured, following drying at 19.5 ◦ C and 45% relative humidity
for 14 days, in an environmentally controlled room.
2.4. Physiological responses
All measurements were conducted with five biological repeats (i.e., 5
plants), following the experimental design. Reported results are aver­
ages ±SD.
2.4.1. Photosynthetic pigments
Concentration of the photosynthetic pigments, chlorophyll a, chlo­
rophyll b, and carotenoids, were measured as previously described
(Saloner et al., 2019). In short, five discs of 0.6 cm in diameter were
dissected from the youngest mature leaf on the main stem after it was
washed twice in distilled water and blotted dry. The pigments were
extracted from the plants following Gorelick et al. (2015), and pigment
concentrations were calculated according to Lichtenthaler and Wellburn
(1983).
2.4.2. Photosynthesis and transpiration
A portable meter, LI-COR 6400XT (LI− COR, Lincoln, NE, USA) was
used to measure photosynthesis rate, transpiration rate and stomatal
conductivity. Water use efficiency (%) (WUE) was calculated as the ratio
between the photosynthesis rate and transpiration rate, multiplied by
100. Measurements were conducted on the youngest mature leaf of the
main stem, at 8–10 AM. In the pruning treatments, the youngest mature
leaf on the highest secondary branch was used for the analysis.
Industrial Crops & Products 167 (2021) 113528
Time of treatment initiation Aim of the treatment
Representing ’natural’ plant development
3 weeks prior to plant maturation Allow better light penetrance and air circulation
3 weeks after the transition to short Directing the plant resources to the top inflorescences, and
day improvement of air circulation as an aid to pest management.
Similar to the defoliation and BBLR Combination of the 2 treatments above
treatments
Conducted every 7 days throughout Development of a large single inflorescence on the main stem,
the growing phase up to 3 weeks aimed at increasing chemical standardization (by reducing
prior to plant maturation variability in micro-climates)
Similar to the 1◦ branch removal Development of several terminal inflorescences, which are
treatment exposed to a similar microclimate, with the goal to increase
chemical standardization
a
Decapitation on day 0 Breaking epical dominance for development of several semi-
main stems, for potentially increasing the number of
inflorescences and hence yield
a
Decapitation on day 0 second Breaking epical dominance for the development of several
pruning at the transition to short day semi-main stems, followed by a second breaking of those semi-
stems’ epical dominance, for development of a highly branched
plant
Photosynthetically active radiation (PAR) was measured near the base of
the plant (Apogee quantum sensor MQ-500 Apogee Instruments, Logan,
UT, USA), to determine the effects of the architectural modulations on
the availability of photosynthetic light.
2.4.3. Membrane leakage
The extent of ion leakage from the cell membrane is considered an
indicator of stress level (Shoresh et al., 2011). The middle leaflet of the
youngest mature leaf on the main stem was used for the analysis, and in
the pruning treatments, the middle leaflet of the youngest mature leaf on
the highest secondary branch was analysed. Measurement and calcula­
tions were conducted following Kravchik and Bernstein (2013); Shoresh
et al. (2011).
2.4.4. Osmotic potential
About 150 mg of leaf tissue from the center of the central leaflet of
the youngest mature leaf on the main stem was used for the analyses. In
the pruning treatments, the center of the central leaflet from the youn­
gest mature leaf on the highest secondary branch was used for the an­
alyses. The measurement was conducted following Shoresh et al. (2011).
2.5. Cannabinoid analyses
To allow evaluation of the effect of the architectural-modulation
treatments, on the standardization of the cannabinoid profile in the
plant, inflorescences were sampled for cannabinoid analyses from five
designated locations in the plants, as is detailed in Table 2. Trimmed
inflorescences were dried at 19.5◦ C and 45% air humidity for 14 days, in
an environmentally-controlled chamber, in the dark. The analysis was
conducted for five replicated plants per treatment, following the
experimental design. For the ’1◦ branch removal’ treatment, which
developed a single inflorescence at the top of the plant, we have eval­
uated spatial standardization within the inflorescent by sampling four
locations: 1-top of the inflorescence; 2 and 3: middle sections from two
opposite sides of the inflorescence; and 4-the bottom part of the
3
N. Danziger and N. Bernstein
Table 2
Locations in the plants sampled for cannabinoid analyses.
No. Location Location adjustments for specific
treatments
1 The apical inflorescences on the For both pruning treatments: The top-
main stem. most apical inflorescence on the plant
2 Apical inflorescence of a secondary For the double-prune treatment: the
branch from the top 1/3 of the plant top most inflorescence of the 2nd or 3rd
branch from the top.
3 Apical inflorescence of a secondary For the double-prune treatment: the
branch from the bottom 1/3 of the top most external inflorescence of the
plant 5th or 6thbranch from the top
4 3◦ inflorescence from an upper 2◦ branch removal: Apical
branch inflorescence from the bottom most
secondary branch
5 3◦ inflorescences from the bottom 2◦ branch removal wasn’t sampled
of the plant- the smallest and least
developed inflorescences were
sampled
inflorescence. For the cannabinoid analysis, the top of the dried in­
florescences was ground using a plastic handheld herb grinder (manu­
facturer unknown). Fifty mg of the ground tissue was placed in a 20 mL
scintillation vial, 10 mL ethanol was added to the tube, and the tube was
shaken in a reciprocal shaker for 1 h at room temperature. The extract
was filtered through a polyvinylidene difluoride (PVDF) membrane fil­
ter of 0.22 μm pore size (Bar-Naor ltd, Ramat Gan, Israel). Cannabinoid
concentrations in the filtered extracts were analyzed using a Jasco 2000
Plus series high performance liquid chromatography (HPLC) system,
which consist of a quaternary pump, autosampler, column compartment
and photodiode array (PDA) detector (Jasco, Tokyo, Japan). The
detection was carried out in a spectrum mode. Chromatographic sepa­
rations were carried out using a Luna Omega 3 μm Polar C18 column
(Phenomenex, Torrance, CA USA) employing acetonitrile:water 75:25
(v/v) with 0.1% (v/v) formic acid, at the isocratic mode, with a flow rate
of 1.0 mL/min. Calculation of cannabinoid concentrations were based
on pure analytical standards that were purchased from Sigma-Aldrich
(Germany): cannabichromene (CBC), cannabichromenic acid (CBCA),
cannabichromevarin (CBCV), cannabigerol (CBG), cannabigerolic acid
(CBGA), cannabinol (CBN), cannabinolic acid (CBNA), cannabidiol
(CBD), cannabidiolic acid (CBDA), cannabicyclol (CBL), cannabidivarin
(CBDV), cannabidivarinic acid (CBDVA), tetrahydrocannabivarinic acid
(THCVA); from Cayman chemical company (Pennsylvania, USA) can­
nabicitran (CBT) and from Restek (Pennsylvania, USA) and tetrahy­
drocannabinolic acid (THCA), Δ9-tetrahydrocannabinol (THC), Δ8-
tetrahydrocannabinol (Δ8-THC), tetrahydrocannabivarin (THCV). R2
values for linear regressions of the calibrations curves of all cannabinoid
standards were >0.994 (Saloner et al., 2021); Concentrations of
Δ8-THC, THCV, CBDV, CBG, CBNA, CBL, CBCV, and CBT, CBN were
lower than the detection limits.
2.6. Evaluation of spatial uniformity (standardization) of the
cannabinoid profile in the plant
Two parameters, ‘Cannabinoid uniformity’ and ’Plant uniformity
score’, were developed to evaluate the effect of the architecture modu­
lation treatments on standardization of the cannabinoid profile in the
plant. The ‘Cannabinoid uniformity’ is a measure of the extent of uni­
formity within the plant of a single cannabinoid. It is the percentage of
samples in a treatment with the concentration of the specific cannabi­
noid within the range ±15% from the treatment-average concentration.
The treatment-average concentration for each cannabinoid was calcu­
lated from the results of the individual samples from the various loca­
tions sampled in each plant per treatment (n=25 per treatment). The
’Plant uniformity score’, is a measure of the integrated uniformity for all
identified cannabinoids within a treatment. It is the average of the ’Plant
uniformity’ scores of all the individual cannabinoids in a treatment.
4
Industrial Crops & Products 167 (2021) 113528
Several concentration margins were tested (±5%, ±10%, ±15%, ±25%,
±50%), resulting in multiple ’Plant uniformity’ scores.
2.7. Statistical analysis
The analysis was performed with the Jump software (version 9, SAS
2015, Cary, NC, USA). The data were subjected to one-way ANOVA
(α<0.05) and a Post-hoc Tukey HSD test was performed for separation of
means. The data met the assumption of homogeneity of variances.
Comparison of relevant means was conducted using Tukey HSD test at α
= 0.05.
3. Results
3.1. Visual appearance of the plants
The visual appearance of the plants differed considerably between
treatments. Images of the plants at maturation, for the two varieties
studied (‘Himalaya’ and ‘Fuji’) are presented in Fig. 1 A–B. The youngest
mature leaf on the main stem (that is considered the representative leaf)
is shown in Fig. 1 supplemental. Following the experimental treatments,
defoliated plants appeared less dense; plants of the ‘BBLR’ and
‘BBLR+Defoliation’ treatments show secondary branching starting
above the bottom 1/3 of the plants; both branch removals treatments
show reduced branching, and both pruning treatments show stimulated
branching and a wider shoot. Both varieties responded similarly to the
treatments (Fig. 1). Leaves of the ‘1◦ branch removal’ treatment were
longer and wider than of all other treatments, and the pruning treat­
ments decreased leaf size (Fig. 1 supplemental).
3.2. Cannabinoid contents
Effects of the plant architecture treatments on cannabinoid concen­
trations in the two varieties are presented in Fig. 2. The results are
concentrations at the apical inflorescence of the main stem [location 1],
which is the conventional representative morpho-developmental tissue
for the analysis of secondary metabolite profile in medical cannabis.
Concentrations of the acidic and decarboxylated forms of the cannabi­
noids were summed, as the latter is a degradation product of the former.
The analysis reveals that the cannabinoid profile is affected by archi­
tectural modulation, and that the sensitivity of the cannabinoid profile
to the architectural treatments vary between genotypes. Specifically, the
chemical profile of’ Fuji’ was more sensitive to the change in plant ar­
chitecture than ‘Himalaya’ (Fig. 2A-B). E.g., while only a few significant
changes in cannabinoid concentrations were detected between treat­
ments in’ Himalaya’, in’ Fuji’, concentrations of most cannabinoids
differed from the control in at least two of the treatments (Fig. 2A-B).
The 1◦ and 2◦ branch removal treatments reduced concentrations of
many of the cannabinoids compared to the ‘Control’. In both varieties,
the ‘1◦ branch removal’ treatment significantly decreased the concen­
trations of all identified cannabinoids (by 25-79%; except for CBCA in’
Fuji’ that increased by 22%), and the ‘2◦ branch removal’, treatment
induced a 11–25% reduction in concentrations for ‘Fuji’, but only 1–8%
in ‘Himalaya’ (except for THC and CBC that were increased by 10–13%).
Interestingly, the defoliation treatments increased CBDVA levels (by
9–19%) while all other treatments had reduced CBDVA by 6–23%. All
treatments (except both branch removal treatments) increased THCA
concentrations (by 5–12%) (Fig. 2A-B).
All cannabinoids are synthesized in the plant in their carboxylated
(acidic) forms, which can later decarboxylate to the non-acidic, bio­
logically active, forms. Very little is known about the effect of cultiva­
tion conditions, and morpho-spatial position in the plant on in-planta
decarboxylation. The physiological state and micro-environments in the
plant, which are altered by the architectural modulation treatments,
may also affect the extent of decarboxylation. Fig. 2C-H present the
concentrations of the acidic and decarboxylated forms of the three
N. Danziger and N. Bernstein
Fig. 1. Effect of the treatments on visual appearance of the plants. ‘Fuji’ variety (A), ’Himalaya’ variety (B). Photographed at the termination of the experiment
during harvest. The treatments are described in Table 1. The bars on the right-hand size represent 1 m. BBLR- Bottom branches and leaves removal.
primary cannabinoids THC, CBD and CBC, and allows to evaluate
treatment effects on decarboxylation. Interestingly, the ratio of the
acidic to the active form was usually specific to a variety by cannabinoid
combination across treatments. For example, for ‘Fuji’ by CBDA
(Fig. 2C) in all treatments, the decarboxylated form was 14–17% out of
the total cannabinoid concentration. Similarly, in ‘Himalaya’ by THCA
(Fig. 2D) 10–13% of the cannabinoid was in the active form. This
demonstrates that the decarboxylation process was insensitive to the
architectural treatments.
Since the supply to patients and to the pharmaceutical industry is
based on inflorescences from the entire plant, ’plant average’ concen­
trations of cannabinoids are of importance. Similar to results for the
apical inflorescence of the main stem, ’plant average’ concentrations
revealed differences between genotypes in sensitivity of the cannabinoid
profile to the plant architecture treatments, with ’Fuji’ being more
sensitive than ’Himalaya’ (Figs. 3 and 4). For example, in ’Himalaya’,
only 2 of the analyzed cannabinoids were affected by more than 2 of the
architecture treatments (P < 0.05) (CBGA, CBDVA), compared with 5 in
’Fuji’ (THCA, THCVA, CBDA, CBDVA, CBGA). Similar to the results for
the apical inflorescence, ‘1◦ branch removal’ had the strongest effect on
the ’plant average’ concentrations as well, followed by ‘2◦ branch
removal’ (Figs. 3 and 4). For both varieties, and all affected cannabi­
noids, these two treatments significantly reduced the detected concen­
trations, except for CBCA that was increased by the ’1◦ branch removal’
treatment in ’Fuji’. These results (Figs. 3 and 4) are in accord with the
treatments’ effects on the concentration of the cannabinoids in the
apical inflorescence (Fig. 2). Furthermore, similar to effects on the apical
inflorescence, both defoliation treatments increased CBDVA concentra­
tions in the ’plant average’ concentration in both varieties (Figs. 3A, 4
A).
Although the trend of the treatment-inflicted changes was similar for
the apical inflorescence and the ’plant average’, the magnitude of the
change was not always the same. For example, the reduction of THCA
concentration by the ’2◦ branch removal’ treatment in ‘Himalaya’ was
not statistically significant in the apical inflorescence but was statisti­
cally significant for the ’plant average’. However, insignificant increase
of CBDA in ‘Fuji’ at the ‘BBLR+Defoliation’ treatment at the apical
inflorescence was amplified and was statistically significant in the plant
average. This suggests as well spatial variability in cannabinoid profiles
throughout the plant.
An important finding is that the ’plant average’ concentration of the
cannabinoids is not identical to concentrations in the apical inflores­
cence. This demonstrates that concentrations throughout the plant are
5
Industrial Crops & Products 167 (2021) 113528
not uniform. For example, concentrations of CBDVA and CBGA in the
apical inflorescence, were higher by 9–23% and 9–20%, respectively
than the ‘plant average’ concentrations, in five treatments in ’Himalaya’
(‘Control’, both pruning treatments, and both defoliation treatments).
The difference in the extent of change in concentration between the
plant average and the apical inflorescence for different treatments sug­
gests treatment effect on the degree of standardization.
For a closer look at the source of the difference between the apical
inflorescence and the plant average, cannabinoid concentrations of 3◦
inflorescences from the bottom of the plant [location 5], which differ
from the apical inflorescences more than any other locations in morpho-
spatial position, are presented in Fig. 2S (supplemental). The in­
florescences at this location are 3◦ inflorescences, which are the smallest
and least developed on the plant, and therefore have the highest po­
tential to vary in physiology and metabolism from the apical in­
florescences. Furthermore, their location at the base of the plant implies
different microclimate than the apical inflorescence of the main stem.
Cannabinoid concentrations in these inflorescences were lower than in
the apical inflorescences in both varieties under all treatments (Fig. 2S,
supplemental). The effect of the treatments was more pronounced at the
3◦ inflorescence from the bottom of the plant [location 5]. Cannabinoid
concentrations increased in this location by all treatments compared
with ‘Control, while at the apical inflorescence of the main stem [loca­
tion 1] no such increase was found for most cannabinoids and treat­
ments (Fig. 2). Similar to the apical inflorescence of the main stem, the
ratio between the decarboxylated and the acidic form remained un­
changed by the treatments. For ‘Fuji’, 14.5–18.5% of the THCA and
17–19.5% of the CBCA were decarboxylated, while for ‘Himalaya’,
12.5–15.5% of the THCA and 14.5–16.5% of the CBCA were degraded to
their active form.
3.3. Variability within the plant
To evaluate if the architecture modification treatments affected the
spatial variability of the cannabinoids’ concentration in the plant, we
have developed two uniformity parameters: ‘Cannabinoid uniformity’
analyses the extent of spatial standardization of a single cannabinoid in
the plant, and ’Plant uniformity score’ presents an integrated measure
for standardization of all analyzed cannabinoids in the plant. Both pa­
rameters are measured in percentages, such that the higher the per­
centage, the lower is the spatial variability. Interestingly, the results
demonstrate that the level of spatial variability of an individual canna­
binoid within the plant (expressed as ’Cannabinoid Uniformity’) is
N. Danziger and N. Bernstein
cannabinoid specific. THCA, which is the most abundant cannabinoid in
the studied cultivars, was found to have the highest level of standardi­
zation (in ‘Fuji’) across all treatments, and the least standardized was
CBGA (Table 3). The level of chemical uniformity for individual can­
nabinoids can vary between genotypes. For example, the ‘Cannabinoid
uniformity’ for CBDVA was 32% and 60% in Himalaya and Fuji,
respectively (at the ‘Control’ treatment), but 76% and 44% at the ‘Single
prune’ treatment (Table 3).
A main goal of the present study was to evaluate if increased stan­
dardization of the cannabinoid profile can be achieved by architecture
manipulation treatments. The results indeed reveal treatment effects on
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Industrial Crops & Products 167 (2021) 113528
Fig. 2. Effect of the architecture treatments on
cannabinoid concentrations in the apical inflo­
rescence. Concentration of each cannabinoid
relative to its concentration in the control
‘Himalaya’ (A) and ‘Fuji’ (B). Concentrations of
major cannabinoids and their decarboxylated
form in the apical inflorescence: THCA (Tetra­
hydrocannabinolic acid) and THC (Δ9-tetrahy­
drocannabinol) (C-D); CBCA (cannabichrome
nic acid) and CBC (cannabichromene) (E-F);
CBDA (cannabidiolic acid) and CBD (cannabi­
diol) (G-H). Gray sections in the bar mark the
acidic forms of the cannabinoids and the white
sections mark the decarboxylated forms.
Different lowercase letters across treatments in
the acidic or decarboxylated sections of the
bars, and different caps above the total treat­
ment bars, represent significant differences be­
tween treatments by Tukey HSD test at α = 0.05
(n=5). BBLR- Bottom branches and leaves
removal.
the level of chemical standardization in the plant for individual canna­
binoids. For example, the ‘cannabinoid uniformity’ of CBGA in ‘Hima­
laya’ was significantly lower for the ’Control’ and ‘1◦ branch removal
treatments compared with five other treatments, (Table 3). The highest
level of spatial uniformity in the plant (100% uniformity) was identified
only for THCA in the ‘BBLR’ treatment and only in’ Fuji’ (Table 3).
The ’Plant uniformity’ score reveal effects of the plant architecture
on the level of standardization of the cannabidiome (Table 3). The least
variation in the cannabidiome was identified for the ‘BBLR’ (in ‘Fuji’),
for which 77% of the inflorescences tested were within a ±15% margin
from the treatment average. The order of cannabidiome uniformity in
N. Danziger and N. Bernstein
‘Fuji’ was ‘BBLR’> ‘Double prune’ > ‘Control’ ≈ ‘Defoliation’ ≈
‘BBLR+Defoliation’ ≈ ‘1◦ Branch removal’ ≈ ‘single prune’ > ‘2◦ Branch
removal’. For’ Himalaya’, the most uniform treatment is ‘Single prune’>
‘Defoliation’ ≈ ‘BBLR+Defoliation’ ≈ ‘Double prune’ ≈ ‘BBLR’ >
‘Control’ ≈ ‘1◦ branch removal’ > ‘2◦ Branch removal’. Similar to’ Fuji’,
the most uniform cannabinoid (for most treatments) is THCA. Due to the
nature of the treatment, for the ‘1◦ branch removal’ treatment the uni­
formity parameters are affected by floral development at a single loca­
tion rather than spatial-environmental differences. It is important to
further investigate interrelations between inflorescence morpho-
development and the cannabidiome towards regulation mechanisms of
spatial variability within the inflorescence.
Calculation of the ‘Plant uniformity’ scores under more restrictive
terms (i.e. allowing only ±5% or ±10% deviations from the plant
average) identified ‘Double prune’ as the most uniform treatment (in
‘Fuji’) (Table 3); while under less restrictive terms (i.e. allowing for
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Industrial Crops & Products 167 (2021) 113528
Fig. 3. Effect of the plant architecture treat­
ments on ’plant average’ concentrations of
cannabinoids in the ‘Himalaya’ variety. CBDVA
(cannabidivarinic acid) (A), CBDA (cannabi­
diolic acid) (B), THCA (tetrahydrocannabinolic
acid) (C), CBGA (Cannabigerolic acid) (D),
THCVA (tetrahydrocannabivarinic acid) (E),
CBCA (cannabichromenic acid) (F), THC (can­
nabichromenic acid) (G), CBC (cannabichro­
mene) (H). The results are mean ±SD (n=20-
25). Different lowercase letters above the means
represent significant differences between treat­
ments by Tukey HSD test at α = 0.05. BBLR-
Bottom branches and leaves removal.
±25%, ±50% deviation from the plant average) there are no significant
differences between the treatments. For ‘Himalaya’, some changes in
uniformity rankings were seen under the different acceptance rates
identifying ‘Defoliation’, ‘BBLR’, and both pruning treatments as the
most uniform treatments. Under the least restrictive term of 50%, all
treatments had a perfect score except for ‘Control’ which scored 95%,
and both branch removal treatments that were considerably lower at
76% and 79%.
Under all levels of acceptance,’ Fuji’ was found to be more uniform
than’ Himalaya’. These results demonstrate the importance of setting
guidelines for the extent of chemical variability as a tool for guiding
plant architecture optimization for standardization.
3.4. Biomass and yield
The architecture modulation treatments affected the biomass of the
N. Danziger and N. Bernstein
developed shoot and shoot organs, as well as yield quantity (Fig. 5A-B).
The changes in biomass deposition were less pronounced for ’Himalaya’
than ’Fuji’ (Fig. 5A-B), and were most affected by the ‘1◦ and 2◦ branch
removal’ treatments. In both varieties, the ‘1◦ branch removal’ treat­
ment had the largest effect on shoot and organs growth. It had signifi­
cantly (P < 0.05) reduced shoot weight in ’Himalaya’ by 50%, resulting
in a 98% yield loss, and it reduced shoot weight of ’Fuji’ more than any
other treatment. In both varieties, the ‘2◦ branch removal’ treatment
increased significantly (P < 0.05) leaves and stem biomass, at the
expense of inflorescence weight loss (29% and 59% reduction, in ‘Fuji’
and ‘Himalaya’, respectively). In both varieties all other treatments did
not affect inflorescences fresh weight (P > 0.05). As expected, the
defoliation treatments reduced fan-leaves biomass, which was also
reduced by the ‘Double prune’ treatment.
The effect of the architecture treatments on yield, which in ’drug-
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Industrial Crops & Products 167 (2021) 113528
Fig. 4. Effect of plant architecture modulation
treatments on ’plant average’ concentrations of
cannabinoids in the ‘Fuji’ variety. CBDVA
(cannabidivarinic acid) (A), CBDA (cannabi­
diolic acid) (B), THCA (tetrahydrocannabinolic
acid) (C), CBGA (Cannabigerolic acid) (D),
THCVA (tetrahydrocannabivarinic acid) (E),
CBCA (cannabichromenic acid) (F), THC (can­
nabichromenic acid) (G), CBC (cannabichro­
mene) (H). The results are mean ±SD (n=20-
25). Different lowercase letters above the means
represent significant differences between treat­
ments by Tukey HSD test at α = 0.05. BBLR-
Bottom branches and leaves removal.
type’ (medical) cannabis is the dry biomass of the inflorescence after
removal (trimming) of the protruding sections of the inflorescence
leaves, reflect the inflicted changes of the treatments on biomass. Only
small changes in yield were induced by most plant architecture modu­
lation treatments, with a slight variation between the two varieties
(Fig. 5C-D). For both verities, ‘1◦ branch removal’ greatly reduced yield
quantity, and the ’2◦ branch removal’ treatment has reduced it to a
lesser extent. For ‘Fuji’, the combination of ‘BBLR+Defoliation’ had
caused a slight yet significant yield loss, while all other treatments did
not significantly affect the total weight of the inflorescences.
3.5. Plant growth and development
Plant height was similar for all treatments, except for plants of both
pruning treatments that in both varieties were significantly shorter. The
N. Danziger and N. Bernstein Industrial Crops & Products 167 (2021) 113528

Table 3
Cannabinoid uniformity’ under a 15% level of acceptance for deviation from the plant average, and ’Plant uniformity scores’ for 5%, 10%,15%, 25% and 50% level of acceptance
for the’ Fuji’ and ‘Himalaya’ varieties.
‘Cannabinoid uniformity’ (%) ’Plant uniformity scores’ (%)

CBGA THCA+THC THCVA CBCA+CBC CBDA+CBD CBDVA 5% 10% 15% 25% 50%

Treatment
Fuji
Control 52ab 88b 60a 72b 72ab 60b 24c 45d 67c 92a 99a
Defoliation 48ab 72c 56a 92a 56c 84a 26c 46d 68c 87a 98a
BBLR + Defoliation 40b 84b 72a 76b 72b 68ab 29b 51c 69c 93a 100a
BBLR 32c 100a 64a 92a 88a 88a 25c 56b 77a 92a 99a
1◦ branch removal 47ab 84b 70a 76ab 65bc 58b 25c 45d 67c 85a 96a
2◦ branch removal 60a 80b 70a 70b 60bc 30c 25c 43d 62d 88a 99a
Single prune 32bc 88b 68a 88a 84ab 44bc 25c 53c 67c 87a 97a
Double prune 56a 88b 84a 80ab 80ab 44bc 35a 59a 72b 89a 100a
Himalaya
Control 48c 64b 44a 64a 48b 32c 21c 35c 50c 72d 95b
Defoliation 68a 72ab 48a 68a 76a 60ab 25b 51a 65b 84c 100a
BBLR + Defoliation 60ab 85a 65a 75a 65ab 40bc 19c 48a 65b 93a 100a
BBLR 48bc 72ab 52a 56ab 60ab 72a 25b 42b 60b 87b 99a
1◦ branch removal 45c 15d 35a 30c 45b 45bc 11e 20e 30d 49f 76c
2◦ branch removal 60ab 40c 50a 50b 70a 55ab 15d 31b 45c 64e 79c
Single prune 64ab 84a 56a 68a 64ab 76a 31a 51a 69a 87b 100a
Double prune 60b 84a 60a 72a 60ab 48b 24b 49a 64b 88b 99a
a
Bottom branches and leaves removal [BBLR]; cannabigerolic acid [CBGA]; tetrahydrocannabinolic acid [THCA]; Δ9-tetrahydrocannabinol [THC]; tetrahy­
drocannabivarinic acid [THCVA]; cannabichromenic acid [CBCA]; cannabichromene [CBC]; cannabidiolic acid [CBDA]; cannabidiol [CBD]; cannabidivarinic acid
[CBDVA].
b
Different lowercase letters near the means represent significant differences between treatments along a column, for each variety, by Tukey HSD test; α = 0.05.

Fig. 5. Effect of plant architecture modulation treatments on biomass accumulation in different plant organs. Fresh weight (Fuji-A, Himalaya -B); and “commer­
cially” dried inflorescences weight (Fuji -C, Himalaya -D). The results are means ±SD (n=5). Different lowercase letters above the means represent significant
differences between treatments by Tukey HSD test at α = 0.05. BBLR- Bottom branches and leaves removal.

reduced height was visible early in development, 18 and 28 days after
pruning in ‘Fuji’ and ‘Himalaya’, respectively (Fig. 6A-B). In addition,
for the ‘Himalaya’ variety, plants of the ‘1◦ branch removal’ treatment
were significantly taller than all other treatments (Fig. 6).
The number of branches that developed on the main stem, and the
average internode length were not affected by the treatments, with the
exception of the pruning treatments and ‘1◦ branch removal’ (Fig. 7A-B,
G–H). A small yet significant change (in ‘Fuji’ only) was detected at the
‘2◦ branch removal’ treatment that developed nearly a branch more than
all other treatments (20.2 ± 0.6 vs. 18.8–19.2 ± 0.7, respectively). This

9
N. Danziger and N. Bernstein
Fig. 6. The effect of plant architecture treatments on plant height throughout the experiment in two varieties, Fuji (A) and Himalaya (B). The results are mean ±SD
(n=5). Asterisk above the averages represent a significant difference between treatments at the given day by Tukey HSD test at α = 0.05. BBLR- Bottom branches and
leaves removal.
trend, although not statistically significant, is apparent also for the
‘Himalaya’ verity. In addition, ’mean internode length’ was longer in the
1◦ branch removal plants. The differences in branch length between
treatments were small (Fig. 7C-D) and followed the trend 3rd > 6th >
10th, with the exception of the pruning treatments. Stem diameter was
not affected by the treatments in ‘Himalaya’, while in ‘Fuji’, plants of the
two pruning treatments had narrower stems compared to all other
treatments.
3.6. Physiological response
The ’Himalaya’ variety was physiologically less affected than ’Fuji’
by the architecture-modulating treatments (Fig. 8). The only treatments
that had significant effects on the plant-atmosphere relations parameters
measured are ’Defoliation’ and the 1◦ and 2◦ branch removal treat­
ments. The photosynthetic activity was reduced only by the ‘1◦ branch
removal’ treatment in ’Fuji’ (by 12%) but was not significantly affected
by the treatments in ’Himalaya’ (Fig. 8A-B). In both varieties, stomatal
conductance was increased by ’Defoliation’ (Fig. 8C-D); the transpira­
tion rate was increased by ’Defoliation’ in ‘Himalaya’ only (Fig. 8E-F);
the plants water use efficiency was reduced by the ‘1◦ branch removal’
treatment in ’Fuji’, as well as by ’Defoliation’ in ‘Himalaya’. In addition,
the ‘Himalaya’ verity exhibited higher water use efficiency than ‘Fuji’.
No differences in intercellular CO2 levels were detected between treat­
ments (Data not shown). Osmotic potential of the leaf sap (Fig. 9E), and
membrane leakage levels were similar across all treatments except for
the ‘1◦ branch removal’ treatment that were 2 fold higher (Fig. 9D). The
concentrations of the photosynthetic pigments were not affected by the
‘Defoliation’, ‘BBLR’ and ‘1◦ Branch removal’ treatments (Fig. 9A-C).
Both the ‘BBLR+Defoliation’ and ‘2◦ Branch removal’ treatments
reduced concentrations of the three pigments. Both pruning treatments
had less chlorophyll b compared with the control, but only ‘Double
prune’ had lower carotenoids levels.
3.7. Light intensity at the plant base
Architecture manipulation treatments affect the 3-D structure of the
shoot and thereby the shoot density, and are hence expected to affect
light penetration to lower parts of the plant. For both varieties, three
treatments allowed the most light to reach ground level, ‘1◦ branch
removal’ and both defoliation treatments. A minor yet significant in­
crease in PAR was measured also in the ‘2◦ branch removal’ treatment in
‘Fuji’ and ‘Double prune’ in ‘Himalaya’ (Fig. 10).
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Industrial Crops & Products 167 (2021) 113528
4. Discussion
4.1. Does plant architecture effect cannabinoid concentration and
standardization?
The therapeutic activity of medical cannabis is dependent on a
plethora of biologically active secondary metabolites which are syn­
thesized in the plant (Andre et al., 2016; Russo, 2019). A major chal­
lenge in utilization of cannabis for modern medicine is the lack of
standardization of the therapeutic metabolite concentrations
throughout the plant (Bernstein et al., 2019a, b), and thereby also in the
plant material supplied to patients. Secondary metabolism in plants is
well known to be affected by environmental conditions, including light
(Magagnini et al., 2018), temperature and humidity (Shamshiri et al.,
2018). Light intensity and quality, which are both altered at longitudinal
positions along the shoot by plant architecture due to shading, were
demonstrated to affect biosynthesis and accumulation of secondary
metabolites (Bian et al., 2015; Ouzounis et al., 2015). We have therefore
hypothesized that modulation of plant architecture, which affects
micro-climates in the plant shoot, will impact production of cannabi­
noids, the major therapeutic secondary metabolites in Cannabis sativa, at
locals throughout the plant, and the spatial standardization of their
concentration. The results revealed an interplay between plant archi­
tecture treatments and concentrations of specific cannabinoids, thereby
assessing the hypothesis.
Importantly, while the apical inflorescence and the plant average
concentrations showed but a small response to plant architecture mod­
ulation, the concentrations at the bottom inflorescences, generally
increased by the modulation treatments, diminishing the concentration
gaps between longitudinal locations on the plant, and hereby increasing
uniformity. The increase of standardization was therefore based on
induced effects on the reproductive tissue at the lower parts of the shoot.
Internal variation in chemical profile is common in plants, and several
studies reported on the variability of secondary metabolite contents
between plant organs and tissues (Cheng et al., 2005; Perry et al., 1999;
Skoula et al., 1996) and along the plant (Singh et al., 1989; Fischer et al.,
2011).
Our results demonstrate that it is possible to alter the secondary
metabolite profile in medical cannabis by architecture modulation
(Figs. 2–4). However, it should be noted, that surprisingly, and contrary
to common belief in the cannabis industry, the changes in cannabinoid
concentrations (Fig. 2–4) and yield (Fig. 5C-D) were small (except for
the ‘1◦ branch removal’ treatment that greatly reduced both parame­
ters). Similar results were reported in a study with roses (Pal and
Mahajan, 2017) which showed only a slight increase in flower yield
under moderate compared with intense pruning, and minor changes in
N. Danziger and N. Bernstein Industrial Crops & Products 167 (2021) 113528

Fig. 7. Morphological changes as effected by plant architecture treatments in two genotypes. Branch number (A- Fuji, B-Himalaya); 3rd, 6th & 10th branch length
(C- Fuji, D- Himalaya); stem diameter (E- Fuji, F- Himalaya); mean internode length (G- Fuji, H- Himalaya). The results are mean ±SD (n=5). Different lowercase
letters above the means represent significant differences between treatments by Tukey HSD test at α = 0.05. BBLR- Bottom branches and leaves removal.

volatiles composition between the two treatments, and no changes in
essential oil content. The quality and quantity of the essential oil was
affected also by the timing and level of pruning (Pal et al., 2014). Diverse
responses to architectural modulation were reported, and in coriander
for example, inflorescence pruning did not affect phenols, flavonoids
and carotenoids contents, while leaves yield, and harvest index were
increased (Campos et al., 2019).
A high score of plant uniformity (>95%), was achieved by all
treatments only if the acceptance level was allowed to increase to ±50%
deviation from the plant average. Under lower acceptance levels, which
allow smaller deviation from the plant average concentration (as is ex­
pected from a medical plant material), a considerably lower values of
‘Plant uniformity score’ were obtained (Table 3). This means, that under
conventional growing conditions the grower will produce a medical
cannabis plant product that contain a very large chemical variability,
±50% of the plant average concentration. This finding is alarming and
should be further studied in light of the potential significant effects on
the therapeutic qualities of the largely unstandardized medical product.
An important finding of the study is that under an acceptance level of
±15% variability from the plant average, which was the acceptance
level used in the present study for most intra-treatment comparison, all
treatments except 1◦ and 2◦ branch removals, increased cannabinoid
uniformity. It should be noted that the treatment-induced changes in
some cannabinoids’ concentration, although significant, were low
(Figs. 2–4) and the practical potential of architectural treatments for
alteration of the chemical profile should be further explored.

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N. Danziger and N. Bernstein Industrial Crops & Products 167 (2021) 113528

Fig. 8. The effect of plant architecture treatments on plant-atmosphere relations parameters: photosynthesis rate (A- Fuji, B-Himalaya); stomatal conductance (C-
Fuji, D- Himalaya); transpiration rate (E- Fuji, F- Himalaya); Water Use Efficiency (G- Fuji, H- Himalaya). The results are mean ±SD of (n=5). Different lowercase
letters above the means represent significant differences between treatments by Tukey HSD test (α = 0.05). BBLR- Bottom branches and leaves removal.

The variability in concentrations of cannabinoids along the plant axis
might reflect endogenous factors such as genetic variability in
biochemical characteristics between developmentally different tissues
(e.g., inflorescences of different ages or of different developmental order
along the stem/branches); exogenous factors (Barra, 2009), such as a
response to different micro-climates within the shoot (e.g., relative hu­
midity, temperature, light) between locations along the plant axis; or
most likely a combination of both. Differences in chemical properties
due to positional factors were found in some species (Bhakta and Gan­
jewala, 2009; Fischer et al., 2011; Rohloff, 1999; Singh et al., 1989) but
not in others (Guitton et al., 2010). While microclimatic changes (e.g.,
temperature and light intensity) have been shown to induce changes in
essential oil quality in lavender (Lavandula angustifolia L.) (Hassiotis
et al., 2014), rosemary (Rosmarinus officinalis L.) (Raffo et al., 2020) and
peppermint (Mentha piperita L.) (Mousavinik et al., 2016). Develop­
mental differences are considered to be spatially-oriented as they are
affected by position and age resulting in a change in size and maturity, as
was shown for lantana (Bhakta and Ganjewala, 2009) and basil (Fischer
et al., 2011).
In our study, the increased uniformity achieved by most treatments
compared with the ‘Control’ is a result of the induced increase in
cannabinoid concentration at the lower parts of the plants, as demon­
strated for 3◦ inflorescences from the bottom of the plant [location 5]
(Fig. 2 supplemental). As the concentration of secondary metabolites are
lower at the bottom part of the shoot compared with the top, raising the
lower concentrations at the bottom reduces the longitudinal gradient

12
N. Danziger and N. Bernstein Industrial Crops & Products 167 (2021) 113528

Fig. 9. Effects of the plant architecture modulation treatments on concentrations of the photosynthetic pigments chlorophyll a (A), chlorophyll b (B) and carotenoids
(C); and cellular response: ion leakage from membranes (D), osmotic potential (E). The results are means ±SD (n=5). Different lowercase letters above the means
represent significant differences between treatments by Tukey HSD test; α = 0.05. BBLR- Bottom branches and leaves removal.

Fig. 10. Effect of plant architecture on light penetration to the base of the plant in two medical cannabis genotypes: Fuji (A), Himalaya (B). The results are means
±SD (n=5). Different lowercase letters above the means represent significant differences between treatments by Tukey HSD test at α = 0.05. BBLR- Bottom branches
and leaves removal.

and increases uniformity. To test the potential role of each component (i. one of these components were compared. i) The ‘Control’ and ‘Defoli­
e., micro-climate and developmental/positional effects) on the observed ation’ treatments are structurally identical, as no branches were
variability in chemical uniformity, two sets of treatments differing in removed, and are therefore suited to examine micro-climate changes

13
N. Danziger and N. Bernstein
effect of defoliation, and so does the comparison of the ‘BBLR’ and
‘BBLR+Defoliation’ treatments. ii) The ‘Control’ and ‘BBLR’ treatments
are developmentally identical at the top 2/3 of the plant, since the leaves
and the secondary branches were removed only after the termination of
the vegetative growth, and as such, they are suited to test developmental
effects as is determined by location (e.g., the developmental locations of
the 3◦ inflorescence from an upper branch [locations 3] and the 3◦
inflorescence from the bottom of the plant [location 5] were located
higher on the plant in the BBLRs treatments), and the same applies for
the ‘Defoliation’ and ‘BBLR+Defoliation’ treatments. Results of this
double test (Table 4) reveal that for ‘Himalaya’, environmental effects
had a higher impact to improve uniformity than developmental stage
effects. The increase in uniformity achieved by defoliation can result
from the increased light uniformity due to penetration to the bottom
parts of the plants (Fig. 10). Unlike ‘Himalaya’, for ‘Fuji’ the develop­
mental factor proved to be more significant. The decline in uniformity
seen by the climatic effects in ‘Fuji’ (Table 4) might be attributed to a
stress response of the defoliated plants (Shulaev et al., 2008) as the ‘Fuji’
variety was more sensitive to the treatments. As the response of the two
cultivars varied, it is important to further investigate if there is a cor­
relation between the intensity of the plants’ physiological response and
the gradient of chemical variance in additional varieties.
4.2. Plant development and function
Two main factors were identified as key to the response of the plants
to the architectural modulation treatments: i. the extent of removal of
meristems or potential sites for reproductive meristem development,
and ii. the developmental stage of the plant at the time of biomass
removal. Yield quantity was reduced only by treatments that involved
extensive removal of potential sites for inflorescence development, and
at late stages of plant development. Accordingly, the most substantial
yield losses, > 90% and 50% reduction, occurred following removal of
1◦ and 2◦ branches, respectively, up to late stages of the flowering
period, keeping only a small number of inflorescences on the plants at
the time of maturation. Yield reduction by architecture manipulation
could result from either a loss of reproductive meristems or from a
reduction in photosynthesizing leaf area and hence carbon fixation. In
cannabis, under flowering indicative conditions, vegetative meristems
switch to reproductive growth and develop inflorescences. Removal of
1◦ and 2◦ branches late in development, removed much of the devel­
oping reproductive meristems resulting in the drastic drop in yield.
Similarly in tomato) Solanum lycopersicum L.), excessive removal of
major branches had a considerable impact on reproduction (Alam et al.,
2016), and in melon (Cucumis melo L.), Silva et al. (2019) showed that
fruit thinning resulted in higher weight per fruit, but lower overall
productivity. Development of optimal architectural manipulation
Table 4
Analysis of differences in ‘Plant uniformity scores’ between pairs of treatments
which are similar in structure, for the evaluation if the source of chemical
variability is related to ‘Developmental’ (positional) or to ‘micro-climate’ ef­
fects. Presented data are averages of differences in ‘Plant uniformity scores’
between the two treatments. (-/+) sign signify decrease/increase of ‘Plant uni­
formity score’.
Difference in ‘Plant
uniformity score’Between
Effects Compared treatments treatments a (%)
‘Fuji’ ‘Himalaya’
BBLR BBLR+Defoliation − 1.4 2.4
‘Micro-climate’
Control Defoliation − 0.2 10.4
Control BBLR 4.4 8 Photosynthetic pigments are therefore not a suitable parameter to assess
‘Developmental’
Defoliation BBLR+Defoliation 3.4 0 effects on chemical quality in cannabis.
a
Calculated as the average of the sum of differences between the two treatments
for the sensitivity levels of 5%, 10%, 15%, 25% and 50%.
b
BBLR- Bottom branches and leaves removal.
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Industrial Crops & Products 167 (2021) 113528
procedures for cannabis cultivation should therefore take into account
the developmental progression following the switch from vegetative to
the reproductive phase, as well as the endogenous shoot morphology.
Other treatments involving removal of meristems that did not in­
fluence the inflorescence yield, were both pruning treatments, and
bottom branches and leaf removal treatment. The main difference be­
tween these treatments and the yield–reducing treatments is the
implementation time. Treatments which involved meristem removal
late in development, did not allow sufficient time for compensative
reproductive growth prior to plant maturation, and had a larger toll on
yield. Therefore, the pruning treatments that were executed early in
plant development (during the vegetative period) did not affect the
reproductive yield; the ‘BBLR’ treatments which were applied 2 weeks
after flowering initiation had an intermediate effect on the reproductive
yield (and in one variety only); while the branch removal treatments
that were conducted weekly well into the flowering phase, reduced yield
considerably. Unlike the lack of effect of pruning on yield in the medical
cannabis studied here, in a field study conducted with hemp cultivars,
pruning once early after flower initiation, have increased seed yield
(Kocjan et al., 2019). This difference might be attributed to the
high-density low-branching agro-technique used in the hemp industry,
hence pruning increased branching and thereby also the number of the
reproductive meristems. These results demonstrate an interaction be­
tween the extent and timing of architecture manipulation and
morphological development, to the compensation ability of meristems
loss in cannabis.
The second reason for a reduction in yield may stem from low carbon
assimilation, which might result from several factors including reduced
light interception, photosynthetic area, assimilation rate and intra-
cellular CO2 as a result of lower transpiration rate, which are expected
to be affected differently by the architecture manipulation treatments.
Light interception by the shoot was diminished by three treatments
(Fig. 10) ‘1◦ Branch removal’ and both defoliation treatments. Similar to
our results for ‘1◦ Branch removal’, in other plants as well, including
cotton (Board et al., 1992) and corn (Torres et al., 2017), lower light
interception resulted in lower yields. However, no such reduction in
yield occurred in the defoliation treatments. Since the defoliation was
conducted late in plant development, light interception by the defoliated
plants was similar to their non-defoliated ‘Control’ and ‘BBLR’ coun­
terparts throughout most of the growing season, while in ‘1◦ Branch
removal’ it was reduced throughout the growing season. A single
treatment had lower photosynthesis rate- ‘1◦ Branch removal’ in ‘Fuji’
(Fig. 8), implying that plants of this treatment not only intercepted less
light, but also used it less effectively. Lower photosynthetic rate is long
known to reduce yield (Zelitch, 1982), further supporting the loss of
yield in the ‘1◦ Branch removal’ treatment. The increase in transpiration
rate per unit leaf area by the defoliation treatments (Fig. 8), reduced
water use efficiency per leaf area (Fig. 8), but since these plants have
much lower leaf area, the increase in water demand per leaf area is
misleading. Moreover, the CO2 concentration within the mesophyll
layer was similar in all treatments, suggesting that photosynthetic effi­
ciency was not affected by carbon availability.
Chemical quality properties of cannabis are derived from the sec­
ondary metabolites, cannabinoids, terpenes and flavonoids in the in­
florescences. For other crops such as mango (Kumar et al., 2020) and
tomato (Ilahy et al., 2018) color, or pigment concentrations, is a key
quality parameter. To test whether there is a correlation between the
accumulation of cannabinoids and other phytochemicals, we tested
treatment effects on the photosynthetic pigments chlorophyll a and b
and carotenoids. The results show that some treatments reduced
pigment accumulation but with no correlation to cannabinoids contents.
Various biotic and abiotic stresses, including excessive light (Vicente
and Boscaiu, 2018) are well-known to elicit secondary metabolites
biosynthesis. Elicitation of stress conditions, or eliciting a stress response
N. Danziger and N. Bernstein
is therefore used for manipulation of secondary metabolite production
(Gorelick et al., 2015; Gorelick and Bernstein, 2014; Ramakrishna and
Ravishankar, 2011; Sathiyabama et al., 2016). In this study, membrane
leakage, an indicator of plant stress (Shoresh et al., 2011), was increased
only in the ‘1◦ Branch removal’ treatment, which reduced chemical
quality. It is possible that this drastic treatment induced a high level of
stress, which combined with the suppressed carbon fixation resulted in
inhibition of secondary metabolism. At our level of knowledge, it cannot
be ruled out that moderate stress could lead to improved quality.
Secondary metabolite production in cannabis is very high compared
to other plants, comprising up to 16% of dry weight in this experiment,
which is equivalent to 3.2% of the fresh weight, compared to 0.046% in
roses (Pal and Mahajan, 2017), 0.4% in sage (Rioba et al., 2015) and
0.8% is sweet basil (Bernstein et al., 2010; Hussain et al., 2008). The
biosynthesis of secondary metabolites is energy consuming and was
likely to reduce due to defoliation and the loss of photosynthetic leaf
area, but since no increase in inflorescence leaves was seen, nor reduc­
tion in yield (Fig. 5C-D), the energy demand for the biosynthesis of the
cannabinoids was satisfied by the existing inflorescence leaves and the
photosynthetic tissues in the flowers. Alternatively, the required energy
or substrates could have been already stored in the plants’ stems or
flowers as sugars or fatty acids at earlier stages of development, prior to
the defoliation, and this issue and other aspects of sink-source relations
in cannabis physiology should be further looked into.
The extent of the treatment-induced changes, which were small in
the present study, might be attributed to the fact that the plants were all
grown to a final height of about 1 m, whereas in the industry the final
size ranges from 50 cm to over 3 m. In larger plants the microclimate,
hormonal, physiological and developmental differences would be bigger
and may increase variation across the plant. For example, in lavender,
variation between locations along the plants in essential oil contents and
composition were observed between plants harvested after reaching
different heights (Dudai et al., 1999), and at least 11 variables that are
affected by plant size were detected to effect peach quality (Génard and
Bruchou, 1992).
5. Conclusions
The results of the present study validate the potential of plant
architectural modulation for regulation of the secondary metabolite
profile in medical cannabis. The level of standardization of most can­
nabinoids in the plants was increased by the treatments by raising the
concentrations at the lower parts of the plant where concentrations are
naturally considerably lower than at the plant top. The treatments that
increased the most the concentrations of the lower inflorescences thus
increasing standardization were ‘Double prune’, and the removal of
bottom branches and leaves. The architecture modulation treatments
elicited cultivar-specific effects on the cannabinoid profile. The
treatment-induced changes in cannabinoid concentrations albeit statis­
tically significant, were small, and although reduced the spatial vari­
ability in the plant, did not completely overcome it. Some cultivar
response variation was seen, pointing at the potential to improve these
traits by classic or genetic breeding for the development of more
chemically uniform cultivars.
CRediT authorship contribution statement
Nirit Bernstein: Conceptualization, Methodology, Supervision,
Writing- Reviewing and Editing. Nadav Danziger: Conducting the ex­
periments, Data analyses, Writing- Original draft preparation.
Declaration of Competing Interest
The authors declare that they have no known competing interests.
15
Industrial Crops & Products 167 (2021) 113528
Acknowledgments
This work was funded by the Chief scientist fund of the Israeli Min­
istry of Agriculture, Grant No. 20-03-0049. The study was conducted at
the commercial farm of Canndoc LTD, a certified commercial cultivation
farm in Israel. We are grateful for the cooperation of Neri Barak and
Adriana Kama from Canndoc LTD. We thank Yael Sade for guidance
concerning the cannabinoid analyses and for technical support; Avia
Saloner, Sivan Shiponi, Dalit Morad and Jecki Shoef for technical sup­
port, and Dr. Uri Hochberg for advice concerning the Licor
measurements.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2021.113528.
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