Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47

Contents lists available at ScienceDirect

Journal of Applied Research on Medicinal and

Aromatic Plants
journal homepage: www.elsevier.com/locate/jarmap

Light dependence of photosynthesis and water vapor

exchange characteristics in different high 9 -THC yielding
varieties of Cannabis sativa L.
Suman Chandra a,∗ , Hemant Lata a , Zlatko Mehmedic a , Ikhlas A. Khan a,b ,
Mahmoud A. ElSohly a,c
a
National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA
b
Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA
c
Department of Pharmaceutics, School of Pharmacy, University of Mississippi, University, MS 38677, USA

a r t i c l e i n f o a b s t r a c t

Article history: The effect of different levels of photon flux densities (000, 400, 800, 1200, 1600
Received 6 January 2015 and 2000 ␮mol m−2 s−1 ) on gas and water vapor characteristics of four high 9 -
Received in revised form 13 February 2015 tetrahydrocannabinol (9 -THC) yielding drug type varieties (HPM, K2, MX and W1) of
Accepted 2 March 2015
Cannabis sativa was studied. Plants of each variety were grown from seeds. On flowering,
Available online 23 March 2015
male plants were removed and vegetatively propagated clones of selected female plants
were used for gas and water vapor studies at different photosynthetic photon flux densi-
Keywords:
ties (PPFDs). Our data show an increasing trend in photosynthesis (PN ), transpiration (E)
Cannabis sativa
and stomatal conductance (gCO2 ) with increase in PPFD up to 2000 ␮mol m−2 s−1 in all
Internal CO2 concentration
Photon flux densities varieties at optimum growth temperature (25 ± 3 ◦ C). However, the magnitude of increase
Photosynthesis and maximum rate of PN (PN max ) varied considerably with the varieties. Highest PN was
Pigment content observed in W1 followed by MX, K2 and HPM. Water use efficiency (WUE) in W1, MX and
Water use efficiency HPM increased with PPFDs up to the highest level tested, whereas, in K2 the highest WUE
was observed at 1600 ␮mol m−2 s−1 . Our results suggest that this species is able to use high
level of PPFDs for its PN and therefore, may be cultivated in sun exposed areas in the field or
under high PPFDs using indoor grow lights for the optimum growth. Strict control of other
environmental factors, however, needs to be maintained while growing the plants indoor.
© 2015 Elsevier GmbH. All rights reserved.

1. Introduction ductivity and yield with their photosynthetic rate, in the

given environment, as more than 90% of dry matter of
Photosynthesis, being the primary source of carbon and live plants is derived from photosynthetic CO2 assimila-
energy, plays a prominent role in plant growth and devel- tion (Zelitch, 1975). Therefore, photosynthesis is a valuable
opment. Generally, the variation in the photosynthetic physiological tool to evaluate the response of plants to
rates of any plant species in different climatic conditions environmental stresses and for the rapid selection of plants
reflects its adjustments to the respective growth environ- for a particular environmental condition (Chandra et al.,
ment and also the resistance to climatic rigors of any kind. 2010a, 2010b; Joshi and Palni, 2005; Monclus et al., 2006)
A close correlation has been reported between plants pro- or selection of suitable indoor growth environmental for a
particular plant species.
In the natural environment, light is an important factor
∗ Corresponding author. Tel.: +1 6629156954; fax: +1 6629155587. affecting developmental, morphological and physiologi-
E-mail address: [email protected] (S. Chandra). cal attributes of individual leaves such as leaf thickness,

http://dx.doi.org/10.1016/j.jarmap.2015.03.002
2214-7861/© 2015 Elsevier GmbH. All rights reserved.
40 S. Chandra et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47

nitrogen fixation per unit area, chlorophyll per unit area, gas and water vapor characteristics on a single C. sativa
and net photosynthesis (Bjorkman, 1981; Boardman, 1997; variety (Chandra et al., 2008). However, C. sativa is an
Ellsworth and Reich, 1993; Hirose and Werger, 1987). Other extremely variable species in terms of morphology, eco-
than photosynthesis, variation in light environment is also physiology and chemical contents due to its allogamous
reported to affect germination, growth and reproduction nature (ElSohly and Slade, 2005; Upton et al., 2013). In
of the plant species (Takahashi and Rustandi, 2006). Leaf many plants species, genotypic and/or clonal variations
photosynthetic response to incident irradiance has been in photosynthetic characteristics and yield in relation to
described by many models and equations (Abreu et al., variable environmental factors have been well reported
2014; Chen et al., 2013; Ozturk et al., 2012; Ruban and (Isebrands et al., 1988; Janssen et al., 1995; Joshi and
Belgio, 2014; Thornley, 1976; Von Caemmerer, 2000). The Palni, 1998). Such information, despite of its high medicinal
capacity of plants to intercept and use radiant energy for and economical values, is completely lacking for C. sativa
photosynthesis determines the availability of photosyn- varieties/clones. Therefore, the present investigation was
thates for growth. However, radiation is not only a source undertaken to determine the variations in photosynthetic
of energy but it is also a stimulus, governing development characteristics among different C. sativa varieties, if any, in
and occasionally stress to the plants (Larcher, 2003). Light relation to light. In this study, variations in gas and water
absorbed in excess of a plant’s photosynthetic capacity is vapor exchange characteristics among four different drug
harmful, creating high light (HL) stress that can lead to pho- type C. sativa varieties (HPM, K2, MX and W1), collected
toinhibition and photodamage. Excitation energy that is from different agro-climatic zones worldwide, at different
not used for photochemistry and not dissipated as fluo- (000, 400, 800, 1200, 1600 and 2000 ␮mol m−2 s−1 ) light
rescence or heat can be transferred to molecular oxygen, levels were studied. The specific objectives of the study
creating highly damaging reactive oxygen species (ROS) were: (1) to evaluate the effect of different light levels on
(Apel and Hirt, 2004; Foyer et al., 1994; Niyogi, 1999). photosynthetic efficiency in C. sativa varieties and (2) to
Therefore, optimum growth environment for photosynthe- explore physiological parameters as a tool for the selection
sis is an important factor which consequently affects the of suitable and optimum light environment for the indoor
physiology, growth and yield of plants. cultivation of these varieties.
Cannabis sativa L. (Cannabaceae) is a widely distributed
plant around the world. It has a long history of medicinal 2. Materials and methods
use with references as far back as the 6th century B.C. C.
sativa is the natural source of cannabinoids (phytocannabi- Plants of four drug type C. sativa varieties i.e. HPM (High
noids), a unique group of terpeno-phenolic compounds Potency Mexican Variety), K2, MX and W1 (all from Terbag
that mainly accumulates in glandular trichomes of the GMBH, Subingen, Switzerland) were grown from seeds in
plant. 9 -Tetrahydrocannabinolic acid (9 -THCA), is the the climatic controlled (25 ± 3 ◦ C temperature, 55 ± 5% RH
precursor of the primary psychoactive agent in C. sativa. and 700 ± 24 ␮mol m−2 s−1 at plant canopy level) indoor
This compound produced as an acid in glandular tri- growing facility at the University of Mississippi. Since
chomes of inflorescence bracts, undergoes decarboxylation female plants of this species contain higher concentration
with age or heating to 9 -tetarhydrocannabinol (9 -THC, of THC and to avoid cross fertilization among the vari-
Fig. 1) (Pertwee, 2006). Besides its psychoactivity, 9 -THC eties, male plants were removed immediately after onset
possesses analgesic, anti-inflammatory, appetite stimu- of flowering. Among the female plants, high 9 -THC yield-
lant and anti-emetic properties making this cannabinoid ing clones were screened for each variety using GC-FID (gas
a promising drug for therapeutic purposes (Sirikantaramas chromatography-flame ionization detector) following Ross
et al., 2007). The pharmacologic and therapeutic potency of et al. (1996) and used for further studies.
C. sativa preparations and 9 -THC have been extensively
reviewed (Brenneisen et al., 1996; Grinspoon and Bakalar, 2.1. Cannabinoids analysis
1993; Mattes et al., 1994). A total of 545 constituents and
104 phytocannabinoids have been isolated from C. sativa Biomass samples taken from apical segments (flow-
so far (ElSohly and Gul, 2014). In view of these medici- ering buds) of different of C. sativa varieties were used
nal properties, research is in progress worldwide to screen for cannabinoids analysis. Samples were dried at 49 ◦ C
and select high THC yielding varieties/clones of C. sativa and individually manicured by hand. Triplicate of each
and to optimize suitable propagation techniques for their sample were used for the cannabinoids analysis. Fol-
efficient conservation and mass-multiplication (Chandra lowing Ross et al. (1996), triplicated 0.1 g samples were
et al., 2008, 2010a, 2010b, 2011a, 2011b, 2013a, 2013b; each extracted with 3 mL of internal standard/extracting
Lata et al., 2009a, 2009b, 2010a, 2010b, 2011, 2012). How- solution (ISTD, 100 mg of 4-androstene-3,17-dione + 10 mL
ever, due to the allogamous (cross fertilization) nature of chloroform + 90 mL methanol) at room temperature for
the species, it is difficult to maintain the chemical pro- 1 h. The extracts were withdrawn into disposable transfer
file of selected high THC-producing genotypes under field pipettes through cotton plugs for filtration and are trans-
conditions. Therefore, indoor cultivation of a selected high ferred into GC vials, which are then capped and placed, on
yielding genotype/clone under controlled environmental the auto sampler. One ␮L aliquots were injected. Six major
conditions is the most suitable way to maintain its potency cannabinoids content 9 -THC, THCV, CBD, CBC, CBG and
and chemical profile. CBN were identified and quantified using Gas chromatog-
In our previous work, we have reported the effect of raphy analysis (GC-FID). Gas chromatography (GC) analysis
environmental variations (CO2 , light and temperature) on was performed using Varian CP-3380 gas chromatograph
 S. Chandra et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47 41

Fig. 1. Chemical structures of major cannabinoids in Cannabis sativa L. 9 -tetrahydrocannabinol (9 -THC), tetrahydrocannabivarin (THCV), cannabidiol
(CBD), cannabichromene (CBC), cannabigerol (CBG) and cannabinol (CBN).

equipped with a Varian CP-8400 automatic liquid sampler, highest THC yielding mother plants of each variety were
a capillary injector and dual flame ionization detectors. selected and used for the photosynthetic measurements.
A 15 m × 0.25 mm DB-1, 0.25 ␮ film (J&W Scientific, Inc.) Throughout the study, all the plants were kept under
column was used. Helium was used as the carrier gas. strict controlled environmental conditions (25 ± 3 ◦ C tem-
An indicating moisture trap and an indicating oxygen perature and 55 ± 5% RH). Indoor light (18 h photoperiod
trap located in the helium line from upstream to down- for vegetative growth and 12 h photoperiod for flowering
stream, respectively, were used. Helium was used as the growth, 700 ± 24 ␮mol m−2 s−1 at plant canopy level, mea-
“make-up” gas at the detector. Hydrogen and compressed sured by LI-COR quantum meter, model LI-189, Lincoln,
air were used as the combustion gases. The instrument Nebraska, USA) was provided with seven full spectrum
parameters used for monitoring samples are: air – 30 psi 1000 W HID (high intensity discharge) lamps in combina-
(400 mL min−1 ), hydrogen – 30 psi (30 mL min−1 ), column tion with seven 1000 W high pressure sodium bulbs (Sun
head pressure – 14 psi (1.0 mL min−1 ), split flow rate – Systems, CA), hung above the plants covering 335 square
50 mL min−1 , split ratio – 50:1, septum purge flow rate – foot area. A hot air suction fan was attached about 3–4 feet
5 mL min−1 , make up gas pressure – 20 psi (20 mL min−1 ), distance between the plants and the bulb to minimize heat-
injector temp – 240 ◦ C, detector temp – 260 ◦ C, initial oven ing due to the HID bulbs. All plants were grown in the same
temp – 170 ◦ C, initial temperature hold time – 1 min, tem- size pots containing the same mixture of top soil, sand and
perature rate – 10 ◦ C/min, final oven temperature – 250 ◦ C manure and, were watered equally and regularly to main-
and final temperature holds time – 3 min. The concentra- tain identical growing conditions. Five healthy and well
tion of a specific cannabinoid is calculated as follows: established randomly selected female clones from each
 GC area (cannabinoid)  variety were used for the photosynthetic measurements
Cannabinoids (%) = and comparison of pigment content.
GC area (ISTD)
 volume (ISTD) 
× × 100 2.3. Photosynthesis and water vapor exchange
amount (sample) measurements

Photosynthetic measurements were made using

2.2. Plant material for gas and water vapor exchange portable photosynthetic system (LI-6400, LI-COR,
experiments Nebraska, USA) equipped with LED red light emitting
source (16400-02, LI-COR, Lincoln, Nebraska, USA) fixed
Vegetative cuttings made from the selected female on the top of the leaf chamber. Leaves of each C. sativa
plants of each variety at vegetative stage (18 h photoperiod) variety were exposed to 000, 400, 800, 1200, 1600 and
prior to being subjected to flowering (12 h photope- 2000 ␮mol m−2 s−1 photosynthetic photon flux densities
riod) were used for the photosynthetic measurements. (PPFDs) under controlled temperature 25 ± 3 ◦ C, humid-
Based on GC-FID analysis, vegetative cuttings of the three ity (55 ± 5%) and CO2 (350 ± 5 ␮mol mol−1 ) conditions.
42 S. Chandra et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47

Measurements were carried out on five upper undamaged, 30

fully expanded and healthy leaves of each of the five clones
(LSD, P < 0.05)
per variety. The rate of dark respiration was measured by
25
maintaining the leaf at zero irradiance in the leaf chamber.

Net Photosynthesis ( µmol m s )

Since the steady state photosynthesis reaches within

-2 -1
30–45 min, the leaves were kept for about 45–60 min 20
under each set of conditions, before the observations were
recorded. Different parameters viz. photosynthetic rate
15
(PN ), transpirational water loss (E), stomatal conductance
for CO2 (gCO2 ) and intercellular CO2 concentration (Ci )
were measured simultaneously at steady state condition 10 HPM
under controlled light and temperature conditions. Water K2
use efficiency (WUE) was calculated as the ratio of rate of MX
photosynthesis and transpiration and a ratio of Ci and gs 5 W1
(Ci /gCO2 ) was used as an indicator of mesophyll efficiency.
0
2.4. Pigments content

Leaves used for the photosynthetic gas exchange -5

400

800

1200

1600

2000
measurements were harvested afterwards for the deter-
mination of pigments content (chlorophyll a, b and -2 -1
carotenoids) following Holms (1954). Three samples PPFD ( µmol m s )
(50 mg leaf tissue each sample) were collected from each
Fig. 2. Photosynthetic response of four high THC yielding drug type vari-
plant. These samples were individually extracted in 10 mL eties of C. sativa (HPM, K2, MX, W1) to different levels of photosynthetic
of dimethyl sulfoxide solvent incubated at 55–60 ◦ C for photon flux densities. Data represent mean ± SD (n = 25).
4–5 h or till tissue became colorless. The absorbance of the
extracts was measured with a UV/visible spectrophotome-
W1 (10.61 ± 1.33) and K2 (7.94 ± 0.19). Fully developed
ter (Model Cary-50, Varian Inc., Palo Alto, California, USA)
vegetative cuttings of these varieties were used for pho-
at 440.5, 644, and 662 nm, and the concentrations of Chl a,
tosynthesis and water vapor exchange measurements.
Chl b and carotenoids were calculated as follows:

Chl a = (9.78 × A662) − (0.99 × A644)

3.2. Photosynthesis in C. sativa varieties under the
× Volume/1000 × fresh sample weight varying light intensity

Chl b = (21.4 × A644) − (4.65 × A662) An efficient photosynthetic activity is necessary for the
production of higher biomass (Zelitch, 1975) and, among
× Volume/1000 × fresh sample weight
the abiotic factors, light is one of the main component
which strongly influences this activity. Light response
Total chlorophyll = Chl a + Chl b
usually provokes physiological alterations, which is deter-
minant for the optimization of gas exchange. Lower or
Carotenoid = {(4.69 × A440.5) − (Total Chl)} × 0.267 higher PPFD levels can result in photo-inhibition and there-
× Volume/1000 × fresh sample weight fore, inhibits photosynthetic process and consequently
growth and productivity of plants (Kitao et al., 2000; Kull,
Pigment contents are expressed as mg g−1 fresh weight 2002). The effect of varying light intensities on photosyn-
of sample. thetic response of different plant species is described by
several authors (Abreu et al., 2014; Bazzaz and Carlson,
2.5. Statistical analysis 1982; Bjorkman 1981; Chen et al., 2014; Hallik et al., 2012;
Wahidin et al., 2013). In this study, an increasing trend in
Statistical analysis of the data on PN , E, gCO2 , Ci , Ci /Ca , PN was observed with light intensity up to the highest level
WUE and Ci /gCO2 with varying PPFDs in different C. sativa tested (i.e. 2000 ␮mol m−2 s−1 ) in all the C. sativa varieties
varieties was done by ANOVA using SAS software (version (Fig. 2). However, considerable variations in the level of PN
9.3, SAS Institute, Cary, NC, USA). were observed. The values of PN max characterize the behav-
ior of plants in relation to the use of photosynthetically
3. Results and discussion active radiation (PAR, Nakazono et al., 2001). In general, the
higher the value of PN max , the greater the photosynthetic
3.1. GC-FID analysis gain. Plants adapted to higher PAR levels, generally, achieve
greater photosynthetic gains (Larcher, 2000) as compared
Table 1 shows the 9 -THC and other cannabinoids to the plants adapted to shade environment. In this study,
content in the four varieties of C. sativa used in this highest rate of PN (PN max , ␮mol m−2 s−1 ) was observed in
study. Highest concentration of 9 -THC (%) was found W1 variety (25.54 ± 3.78) followed by MX, K2 and HPM
in HPM (12.09 ± 0.65) followed by MX (10.66 ± 1.05), (23.70 ± 2.98, 21.7 ± 2.41 and 19.82 ± 3.12, respectively).
 S. Chandra et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47 43

Table 1
Quantitative determination of 9 -THC and other cannabinoids content (%) in elite female clones of four high 9 -THC yielding varieties of Cannabis sativa
L.

Varieties 9 -THC CBD CBC THCV CBG CBN

HPM 12.09 ± 0.65 0.04 ± 0.02 0.14 ± 0.01 0.08 ± 0.01 0.36 ± 0.09 0.15 ± 0.08
K2 7.94 ± 0.19 0.03 ± 0.02 0.15 ± 0.01 0.05 ± 0.01 0.13 ± 0.01 0.13 ± 0.01
MX 10.66 ± 1.05 0.04 ± 0.02 0.17 ± 0.03 0.05 ± 0.01 0.43 ± 0.01 0.19 ± 0.06
W1 10.61 ± 1.33 0.02 ± 0.01 0.14 ± 0.08 0.08 ± 0.01 0.56 ± 0.03 0.14 ± 0.07

Data represents mean ± SD, n = 3.

Increasing trend in PN with light intensity up to the high- 7

est level tested in these varieties reflects their adaptability
(LSD, P < 0.05)
potential at higher light environment. Similar results were
reported in a previous report by our group on a single C. 6
sativa variety (Chandra et al., 2008).

Transpiration ( mmol m s )
-2 -1
The highest effect of light intensities on PN was observed
in variety HPM followed by K2, MX and W1. The rate of PN 5
was enhanced 140% in HPM and 94%, 71% and 44% in K2, MX
and W1 varieties respectively, when plants were exposed
from 400 to 2000 ␮mol m−2 s−1 PPFD. It was interesting to 4
note that the variety maintaining higher rate of PN at higher HPM
PPFD also maintained high PN at lower light levels and had K2
lower percent increase in PN when exposed from lower 3 MX
to the higher PPFDs. Dark respiration (Rd , 0 ␮mol m−2 s−1 W1
PPFD) was observed highest in W1 (2.70 ± 0.29) followed
by MX, K2 and HPM (2.60 ± 0.31, 2.19 ± 0.29 and 1.81 ± 0.38 2
respectively).

1
0

400

800

1200

1600

2000
3.3. Pigments content.
-2 -1
The pigments content (Chl a, Chl b, total Chl, Chl a/Chl PPFD ( µmol m s )
b ratio, Car and total Chl/Car ratio) in different C. sativa
Fig. 3. Rate of transpiration in different C. sativa varieties to different
varieties are shown in Table 2. The capacity of leaves to pho- levels of photosynthetic photon flux densities. Data represent mean ± SD
tosynthesize varies with the environment in which they (n = 25).
develop (Senser et al., 1975) and the magnitude of pho-
tosynthetic processes also varies with variations in leaf
properties, e.g. leaf chlorophyll contents, chlorophyll a:b
ratio and the carotenoid. These variations in leaf properties differences in pigment content may be due to their basic
are distinct adaptations to the prevailing light environment genetic makeup.
(Chandra and Todaria, 1984). Plants adapted to lower light
environment, generally, have a higher chlorophyll concen-
tration per unit of fresh mass and a lower chlorophyll a/b 3.4. Water vapor exchange characteristics
ratio than sun leaves, because a greater quantity of chloro-
phyll is associated with antennae molecules in shade leaves The effect of PPFDs on transpiration (E) and stomatal
(Craine and Reich, 2005; Evans and Poorter, 2001). These conductance (gCO2 ) are shown in Figs. 3 and 4, respec-
changes maximize light interception and increase carbon tively. Similar to PN , a gradual increase in E and gCO2
gain in low light, through more efficient investment in were observed with increasing PPFDs in all varieties. How-
photosynthetic machinery (Evans and Poorter, 2001). In ever, the magnitude of increase varied among varieties.
the present study, the highest amount of chlorophyll con- About 83% increase in E and ∼107% increase in gCO2
tent (total Chl) was observed in HPM (2.72 ± 0.18 mg g−1 was observed in the HPM variety, whereas, ∼82%, 65%
fresh weight) followed by MX (2.68 ± 0.61 mg g−1 fw), K2 and 53% increase in E and, 57%, 78% and 62% increase
(2.33 ± 0.20 mg g−1 fw) and W1 (1.92 ± 0.14 mg g−1 fw) in gCO2 were observed in K2, MX and W1, respectively,
varieties. when exposed to the highest level (2000 ␮mol m−2 s−1 )
Similarly, carotenoids (Car) content which is reported of PPFD as compared to 400 ␮mol m−2 s−1 . The rate of
to protect leaves from photo-damage under adverse transpiration (mmol m−2 s−1 ) was highest in MX variety
environmental conditions was also highest in HPM (6.62 ± 0.32) followed by W1 (6.59 ± 0.29), K2 (6.20 ± 0.33)
(0.40 ± 0.05 mg g−1 fw) followed by MX (0.39 ± 0.08 mg g−1 and HPM (4.99 ± 0.42) whereas, gCO2 (mmol m−2 s−1 ) was
fw), K2 (0.32 ± 0.04 mg g−1 fw) and W1 (0.25 ± 0.02 mg g−1 highest in MX (256.33 ± 16.64) followed by HPM, K2 and
fw) varieties. Given the facts that these plants were grown W1 with the values of 232.77 ± 18.50, 210.39 ± 17.42 and
under controlled and similar environmental conditions the 163.63 ± 14.52, respectively at 2000 ␮mol m−2 s−1 PPFD.
44 S. Chandra et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47

Table 2
Pigments content (mg g−1 fresh weight) in different drug type varieties of C. sativa L.

Varieties Chl a Chl b Chl a/b ratio Total Chl Car Total Chl/Car

HPM 1.99 ± 0.12 0.73 ± 0.06 2.74 ± 0.05 2.72 ± 0.18 0.40 ± 0.05 6.83 ± 0.42
K2 1.69 ± 0.13 0.64 ± 0.08 2.65 ± 0.15 2.33 ± 0.20 0.32 ± 0.04 7.24 ± 0.42
MX 1.81 ± 0.12 0.87 ± 0.57 2.63 ± 1.26 2.68 ± 0.61 0.39 ± 0.08 6.93 ± 0.55
W1 1.49 ± 0.11 0.43 ± 0.06 3.51 ± 0.49 1.92 ± 0.14 0.25 ± 0.02 7.80 ± 0.53

Chl a, chlorophyll a; Chlb, chlorophyll b; total Chl, total chlorophyll; Chl a/Chl b ratio, chlorophyll a/chlorophyll b; Car, carotenoid; Chl/Car ratio, total
chlorophyll/carotenoid. Data represent mean ± SD, n = 25.

275 400
(LSD, P < 0.05) (LSD, P < 0.05)
250 HPM

Intercellular CO 2 Concentration ( ml L )
HPM

-1
Stomatal Conductance (mmol m s )

K2
-2 -1

225 K2 MX
MX W1
W1
200 300

175

150

125 200

100

75

50 100 0

400

800

1200

1600

2000
0

400

800

1200

1600

2000

-2 -1 -2 -1
PPFD ( µmol m s ) PPFD ( µmol m s )

Fig. 4. Effect of increasing levels of photosynthetic photon flux densities Fig. 5. Intercellular CO2 concentration in different C. sativa varieties to
on stomatal conductance in different C. sativa varieties. Data represent different levels of photosynthetic photon flux densities. Data represent
mean ± SD (n = 25). mean ± SD (n = 25).

3.5. Intercellular CO2 concentration and MX, respectively when exposed to 2000 ␮mol m−2 s−1
PPFDs as compared to 400 ␮mol m−2 s−1 PPFDs. A decrease
Intercellular CO2 concentration (Ci , ␮mol mol−1 ) and in Ci and Ci /Ca ratio with increasing PPFDs under differ-
the ratio of intercellular to ambient CO2 concentration ent temperature conditions (20, 25, 3, 35 and 40 ◦ C) have
(Ci /Ca ) are shown in Figs. 5 and 6, respectively. Intercel- also been reported by our group in a single high potency
lular CO2 concentration (Ci ) in leaf tissue is considered to Mexican C. sativa variety (HPM) (Chandra et al., 2008).
be a set point to determine how plants are responding Although essentially a biochemical process, photosyn-
to the fluctuating environment (Ehleringer and Cerling, thesis is often regarded as a diffusive process. The rate of
1995) and provides a mean to assess the relative impor- diffusion of CO2 between leaf and environment is largely
tance of stomatal and mesophyll processes in controlling controlled by two factors, stomatal conductance (gCO2)
PN (Renburg and Kruger, 1993). Photosynthetic response and CO2 concentration gradient between carboxylation
involves photosynthesis in stomatal guard cells as well site and ambient air (Ca ). This CO2 concentration gradi-
as that in mesophyll cells (Zeiger et al., 2002). Vigor- ent at given gCO2 and Ca is established predominantly by
ous mesophyll photosynthesis lowers the Ci , and the Ci Ci . Further analysis of the photosynthetic data particularly
thus lowered causes stomatal opening (Wang et al., 2008). gCO2 and Ci at different light intensities indicate that the
This has been regarded as a major mechanism of reg- mechanism of control of photosynthesis by light levels
ulation of stomatal aperture by photosynthesis. In the differs with C. sativa varieties. Both stomatal and meso-
present study, all the C. sativa varieties showed a pattern phyll factors seem to be responsible for the differences
of decreasing Ci and Ci /Ca ratio with increase in PPFDs. in light dependence of photosynthesis, however, their
A decrease of about 58%, 51%, 41% and 44% in Ci was magnitude varied with varieties. The observed low pho-
observed in HPM, K2, W1 and MX, respectively due to tosynthetic activity in K2 and HPM varieties as compared
high PPFD (2000 ␮mol m−2 s−1 ) as compared to those at to W1 and MX could be ascribed to their lower Ci values
400 ␮mol m−2 s−1 . Similarly, a decrease of about 55%, 52%, irrespective of the light levels. Sheshshayee et al. (1996)
42% and 45% in Ci /Ca ratio was observed in HPM, K2, W1 have reported Ci /gCO2 ratio as an indicator of mesophyll
 S. Chandra et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47 45

Table 3
Effect of different PPFDs on Ci /gCO2 ratios in C. sativa L. varieties.

Parameter PPFD (␮mol m−2 s−1 ) Cannabis varieties

HPM K2 MX W1

Ci /gCO2 000 (Dark) 3.39 ± 0.13 3.29 ± 0.13 2.90 ± 0.17 4.76 ± 0.14
400 2.14 ± 0.17 1.80 ± 0.14 1.81 ± 0.24 2.78 ± 0.16
800 1.60 ± 0.20 1.50 ± 0.23 1.51 ± 0.24 2.41 ± 0.27
1200 1.38 ± 0.19 1.37 ± 0.11 1.33 ± 0.17 2.00 ± 0.12
1600 1.14 ± 0.15 1.17 ± 0.09 1.06 ± 0.11 1.79 ± 0.20
2000 0.75 ± 0.06 0.81 ± 0.07 0.79 ± 0.04 1.34 ± 0.19

Ci , intercellular CO2 concentration; gCO2 , stomatal conductance for CO2 . Data represent mean ± SD, n = 25.

1.5 6

(LSD, P < 0.05) HPM (LSD, P < 0.05)

K2
MX
4

Water Use Efficiency x 100

W1
1
C i/C a

2 HPM
K2
MX
W1
0.5
0

0 -2
0

400

800

1200

1600

2000

400

800

1200

1600

2000
-2 -1 -2 -1
PPFD ( µmol m s ) PPFD ( µmol m s )

Fig. 6. Effects of different photosynthetic photon flux densities on the Fig. 7. Effects of different photosynthetic photon flux densities on the
ratio of intercellular CO2 (Ci ) concentration to ambient CO2 (Ca ) concen- water use efficiency of different C. sativa varieties. Data represent
tration (Ci /Ca ) in different C. sativa varieties. Data represent mean ± SD mean ± SD (n = 25).
(n = 25).

varieties and a slight decrease in K2 at higher PPFD (i.e.

efficiency and a representation of mesophyll control on PN . 2000 ␮mol m−2 s−1 ). Plants with high rate of PN and WUE
Our data shows an increase in mesophyll efficiency (i.e. are reported to exhibit better potential to grow faster and
decreasing Ci /gCO2 ratio) with increasing PPFDs in all four yield more as compared to the species/varieties with low
C. sativa varieties up to 2000 ␮mol m−2 s−1 PPFDs (Table 3). PN and WUE under fluctuating environmental conditions
A decrease of about 64%, 55%, 56% and 56% in Ci /gCO2 ratio (Jones, 1992; Zhang et al., 1996). Our results show that
was observed in HPM, K2, W1 and MX, respectively due the C. sativa varieties studied have higher PN and WUE
to high PPFD (2000 ␮mol m−2 s−1 ) as compared to those at under the upper range of PPFDs, which reflects their growth
400 ␮mol m−2 s−1 . potential at high light levels.

3.6. Water use efficiency 4. Conclusion

The effect of increasing PPFDs on water use efficiency In conclusion, a considerable variation in light response
(WUE) in C. sativa varieties is shown in Fig. 7. Water use of photosynthesis was observed in different drug type C.
efficiency is an important index revealing the relationship sativa varieties. The higher rates of PN and WUE of W1
between water consumption and biomass production in and MX varieties at higher light levels as compared to K2
plants. At the leaf level, WUE is defined as CO2 uptake by and HPM show their physiological plasticity to acclima-
per unit of transpiration and calculated as a ratio of PN and tize with higher light levels. Since these plants were grown
E. In the present study, WUE in these varieties increased under controlled and identical environmental conditions,
with PPFDs up to its highest level at 1600 ␮mol m−2 s−1 variations in photosynthetic response represent the differ-
followed by a plateau in its values in HPM, MX and W1 ences in their genetic makeup and reflect their inherited
46 S. Chandra et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47

tendency for preferred growth environment. Higher PN in Craine JM, Reich PB, 2005. Leaf-level light compensation points in shade-
tolerant woody seedlings. New Phytologist 166, 705–708.
combination with higher WUE and mesophyll efficiency
Ehleringer JR, Cerling TE, 1995. Atmospheric CO2 and the ratio of intercel-
under the high PPFDs in C. sativa varieties reflects their lular to ambient CO2 concentrations in plants. Tree Physiology 15 (2),
greater adaptability potential to the high light levels. 105–111.
Ellsworth DS, Reich PB, 1993. Canopy structure and vertical patterns of
photosynthesis and related leaf traits in a deciduous forests. Oecologia
Acknowledgment 96, 169–178.
ElSohly MA, Slade D, 2005. Chemical constituents of marijuana: the com-
plex mixture of natural cannabinoids. Life Sciences 78, 539–548.
The work was supported in part with federal funds from ElSohly MA, Gul W, 2014. Constituents of Cannabis sativa. In: Pertwee RG
the National Institute on Drug Abuse (NIDA), National Insti- (Ed.), Handbook of Cannabis. Oxford University Press, pp. 3–22.
Evans R, Poorter H, 2001. Photosynthetic acclimation of plants to growth
tute of Health (NIH), Department of Health and Human
irradiance. Plant, Cell and Environment 24, 755–767.
Services, USA, under the contract No. N01DA-5-7746. Foyer CH, Lelandais M, Kunert KJ, 1994. Photooxidative stress in plants.
Physiologia Plantarum 92, 696–717.
Grinspoon L, Bakalar JB, 1993. Marihuana, the Forbidden Medicine. Yale
References University Press, New Haven.
Hallik L, Niinemets Ü, Kull O, 2012. Photosynthetic acclimation to light in
Abreu PP, Souza MM, Almeida AAF, Santos EA, Freitas JCO, Figueiredo woody and herbaceous species: a comparison of leaf structure, pig-
AL, 2014. Photosynthetic responses of ornamental passion flower ment content and chlorophyll fluorescence characteristics measured
hybrids to varying light intensities. Acta Physiologiae Plantarum 36, in the field. Plant Biology 14, 88–99.
1993–2004. Hirose T, Werger MJA, 1987. Nitrogen use efficiency and daily photosyn-
Apel K, Hirt H, 2004. Reactive oxygen species: metabolism, oxidative thesis of leaves in the canopy of a Solidago altissma stand. Physiologia
stress, and signal transduction. Annual Review of Plant Biology 55, Plantarum 70, 215–222.
373–399. Holm G, 1954. Chlorophyll mutation in barley. Acta Agriculturae Scandi-
Bazzaz FA, Carlson RW, 1982. Photosynthetic acclimation to variability in navica 4, 457–471.
the light environment of early and late successional plants. Oecologia Isebrands JG, Ceulemans R, Wiard B, 1988. Genetic variation in photosyn-
54, 313–316. thetic traits among Populus clones in relation to yield. Plant Physiology
Bjorkman O, 1981. Responses to different quantum flux densities. In: and Biochemistry 26, 427–437.
Lange OL, Nobel PS, Osmond CB, Ziegler H (Eds.), Physiological Plant Janssen LHJ, VanOeveren JC, VanHasselt PR, Kuiper PJC, 1995. Genotypic
Ecology I: Encyclopedia of Plant Physiology. New Series, vol. 12A. variation in chlorophyll fluorescence parameters, photosynthesis and
Springer, Berlin, pp. 57–107. growth of tomato grown at low temperature and low irradiance. Pho-
Boardman NK, 1997. Comparative photosynthesis of sun and shade plants. tosynthetica 31, 301–314.
Annual Review of Plant Physiology 28, 355–377. Jones HG, 1992. Plants and Microclimate: Quantitative Approach to Envi-
Brenneisen R, Egli A, ElSohly MA, Henn V, Spiess Y, 1996. The effect of ronmental Plant Physiology, 2nd ed. Cambridge University Press,
orally and rectally administered D9-tetrahydrocannabinol on spas- Cambridge.
ticity. A pilot study with two patients. International Journal of Clinical Joshi SC, Palni LMS, 1998. Clonal variation in temperature response of
Pharmacology and Therapeutics 34, 446. photosynthesis in tea. Plant Science 137, 225–232.
Chandra P, Todaria NP, 1984. Leaf pigmentation in Berberis species from Joshi SC, Palni LMS, 2005. Greater sensitivity of Hordeum himalayens
different altitudes. Photosynthetica (Prague) 18, 414–417. Schult. to increasing temperature causes reduction in its cultivated
Chandra S, Lata H, Khan IA, ElSohly MA, 2008. Photosynthetic response of area. Current Science 89, 879–882.
Cannabis sativa L. to variations in photosynthetic photon flux densities, Kitao M, Lei TT, Koike T, Tobita H, Maruyama Y, Matsumoto Y, Ang
temperature and CO2 conditions. Physiology and Molecular Biology of LH, 2000. Temperature response and photoinhibition investigated by
Plants 14, 299–306. chlorophyll fluorescence measurements for four distinct species of
Chandra S, Lata H, Mehmedic Z, Khan IA, ElSohly MA, 2010a. Assessment dipterocarp trees. Physiologia Plantarum 109, 284–290.
of cannabinoids content in micropropagated plants of Cannabis sativa Kull O, 2002. Acclimation of photosynthesis in canopies: models and lim-
L. and their comparison with conventionally propagated plants and itations. Oecologia 133, 267–279.
mother plant during developmental stages of growth. Planta Medica Larcher W, 2000. Ecofisiologia vegetal. RIMA Artes e Textos, São Carlos.
76, 743–750. Larcher W, 2003. Physiological Plant Ecology: Ecophysiology and Stress
Chandra S, Lata H, Khan I, ElSohly MA, 2010b. Propagation of elite Cannabis Physiology of Functional Groups.
sativa for the production of 9 -tetrahydrocannabinol (THC) using Lata H, Chandra S, Khan I, ElSohly MA, 2009a. Thidiazuron induced high
biotechnological tools. In: Arora R (Ed.), Medicinal Plant Biotechnol- frequency direct shoot organogenesis of Cannabis sativa L. In Vitro
ogy. CABI-UK, pp. 98–114. Cellular and Developmental Biology-Plant 45, 12–19.
Chandra S, Lata H, Khan IA, ElSohly MA, 2011a. Temperature response of Lata H, Chandra S, Khan I, ElSohly MA, 2009b. Propagation through
photosynthesis in different drug and fiber varieties of Cannabis sativa alginate encapsulation of axillary buds of Cannabis sativa L. – an impor-
L. Physiology and Molecular Biology of Plants 17, 297–303. tant medicinal plant. Physiology and Molecular Biology of Plants 15,
Chandra S, Lata H, Khan IA, ElSohly MA, 2011b. Photosynthetic response 79–86.
of Cannabis sativa L., an important medicinal plant, to elevated levels Lata H, Chandra S, Techen N, Khan I, ElSohly MA, 2010a. Assessment of
of CO2 . Physiology and Molecular Biology of Plants 17 (3), 291–295. genetic stability of micropropagated Cannabis sativa plants by ISSR
Chandra S, Lata H, Khan I, ElSohly MA, 2013a. The role of biotechnology in markers. Planta Medica 76, 97–100.
Cannabis sativa propagation for the production of phytocannabinoids. Lata H, Chandra S, Khan I, ElSohly MA, 2010b. High frequency plant regen-
In: Chandra S, Lata H, Varma A (Eds.), Biotechnology for Medicinal eration from leaf derived callus of high 9 -tetrahydrocannabinol
Plants – Micropropagation and Improvement. Springer-Verlag, Berlin, yielding Cannabis sativa L. Planta Medica 76, 1629–1633.
Heidelberg, pp. 123–148. Lata H, Chandra S, Techen N, Khan I, ElSohly MA, 2011. Molecular analysis
Chandra S, Lata H, Khan I, ElSohly MA, 2013b. Macroscopic identification of genetic fidelity in Cannabis sativa L. plants grown from synthetic
of Cannabis sativa. In: Upton R, Craker L, ElSohly MA, Romm A, Russo E seeds following in vitro storage conditions. Biotechnology Letters 33,
(Eds.), Cannabis Inflorescence and Leaf – Standers of Identity, Analysis 2503–2508.
and Quality Control, American Herbal Pharmacopeia Monograph. , pp. Lata H, Chandra S, Mehmedic Z, Khan I, ElSohly MA, 2012. In vitro
9–13. germplasm conservation of high 9 -tetrahydrocannabinol yielding
Chen JW, Kuang SB, Long GQ, Meng ZG, Li LG, Chen ZJ, Zhang GH, Yang elite clones of Cannabis sativa L. under slow growth conditions. Acta
SC, 2014. Steady-state and dynamic photosynthetic performance and Physiologiae Plantarum 34, 743–750.
nitrogen partitioning in the shade-demanding plant Panax notogin- Mattes RD, Egelman K, Shaw LM, ElSohly MA, 1994. Cannabinoids
seng under different levels of growth irradiance. Acta Physiologiae appetite stimulation. Pharmacology, Biochemistry and Behavior 49,
Plantarum 36, 2409–2420. 187.
Chen JW, Yang ZQ, Zhou P, Hai MR, Tang TX, Liang YL, An TX, 2013. Accumu- Monclus R, Dreyer E, Villar M, Delmotte FM, Delay D, Petit JM, Bar-
lation and partitioning, photosynthesis, and photosynthetic induction baroux C, Thiec DL, Brechet C, Brignolas F, 2006. Impact of drought
in field-grown maize (Zea mays L.) under low- and high-nitrogen con- and productivity and water use efficiency in 29 genotypes of Populus
ditions. Acta Physiologiae Plantarum 35, 95–105. deltoids × Populus nigra. New Phytologist 169, 765–777.
 S. Chandra et al. / Journal of Applied Research on Medicinal and Aromatic Plants 2 (2015) 39–47 47

Nakazono EM, Costa MC, Futatsugi K, Paulilo MTS, 2001. Initial growth Sirikantaramas S, Taura F, Morimoto Y, Shoyama Y, 2007. Recent advances
of Euterpe edulis Mart. in different light regimes. Revista Brasileira de in Cannabis sativa research: biosynthetic studies and its potential in
Botânica 24, 173–179. biotechnology. Current Pharmaceutical Biotechnology 8, 237–243.
Niyogi KK, 1999. Photoprotection revisited: genetic and molecular Takahashi K, Rustandi A, 2006. Responses of crown development to
approaches. Annual Review of Plant Physiology and Plant Molecular canopy openings by saplings of eight tropical submontane forest tree
Biology 50, 333–359. species in Indonesia: a comparison with cool temperate trees. Annals
Ozturk I, Holst N, Ottosen CO, 2012. Simulation of leaf photosynthesis of Botany 97, 559–569.
of C3 plants under fluctuating light and different temperatures. Acta Thornley JHM, 1976. Mathematical Models in Plant Physiology. Academic
Physiologia Plantrum 34, 2319–2329. Press, London.
Pertwee RG, 2006. Cannabinoid pharmacology: the first 66 years. British Upton R, Craker L, ElSohly MA, Romm A, Russo E (Eds.), 2013. Cannabis
Journal of Pharmacology and Chemotherapy 147, 163–171. Inflorescence and Leaf – Standards of Identity, Analysis and Quality
Renburg LV, Kruger GHJ, 1993. Comparative analysis of differential Control, American Herbal Pharmacopeia. , p. 61.
drought stress-induced suppression and recovery in CO2 fixation: sto- Von Caemmerer S, 2000. Biochemical Models of Leaf Photosynthesis.
matal and not stomatal limitation in Nicotiana tabacum L. Journal of CSIRO Publishing, Collingwood, Australia.
Plant Physiology 142, 296–306. Wahidin S, Idris A, Shaleh SRM, 2013. The influence of light intensity
Ross SA, Parker M, Arafat R, Lovett K, ElSohly MA, 1996. The Analysis and photoperiod on the growth and lipid content of microalgae Nan-
of Confiscated Marijuana Samples for Different Cannabinoids Using nochloropsis spp. Bioresource Technology 129, 7–11.
GC/FID. American Laboratory, pp. 16–17. Wang Y, Noguchi K, Terashima I, 2008. Distinct light responses of the adax-
Ruban AV, Belgio E, 2014. The relationship between maximum tol- ial and abaxial stomata in intact leaves of Helianthus annuus L. Plant,
erated light intensity and photoprotective energy dissipation in Cell and Environment 31, 1307–1316.
the photosynthetic antenna: chloroplast gains and losses. Philo- Zeiger E, Talbott LD, Frechilla S, Srivastava A, Zhu J, 2002. The guard cell
sophical Transactions of the Royal Society of London 369, 1471– chloroplast: a perspective for the twenty-first century. New Phytolo-
2970. gist 153, 415–424.
Senser M, Schotz E, Beck E, 1975. Seasonal changes in structure and func- Zelitch I, 1975. Improving the efficiency of photosynthesis. Science 188,
tion of spruce chloroplasts. Planta 126, 1–10. 626–633.
Sheshshayee MS, KrishnaPrasad BT, Natraj KN, Sankar AGP, Udayakumar Zhang JW, Marshall JD, Fins L, 1996. Correlated population differences in
M, 1996. Ratio of intercellular CO2 concentration of mesophyll effi- dry matter accumulation, allocation and water use efficiency in three
ciency. Current Science 70, 672–675. sympatric conifer species. Forensic Science 42, 242–249.