Photographs of Cannabis sativa mother plants just after [A, C; 112 days after planting (DAP)] or just before severance (B, D; 133 DAP) as well as a photograph of an apical stem cutting (E), for genotype ‘Original Blitz’ (A, B) and genotype ‘King Harmony’ (C, D, E).
Fig. 2.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on rooting of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Fraction of rooted cuttings (C, D) and root dry mass (g per cutting) of rooted cuttings. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 3.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on cutting dry mass and leaf area of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Cutting dry mass (g per cutting) (C, D) and leaf area. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 4.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on indole-3-acetic acid (IAA) concentration in organs of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) in apex; (C, D) in leaf; (E, F) and in stem. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 5.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on starch content in organs of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) In apex; (C, D) in leaf; (E, F) and in stem. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 6.
Effects of photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation and propagation on rooting of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Fraction of rooted cuttings (C, D) and root dry mass (g per cutting) of rooted cuttings. Data points indicate means of three blocks each consisting of nine replicate plants. Error bars represent the standard error of means derived from the analysis of variance. Data have been pooled for mother plant age 65 and 156 DAP. P values, when significant, are shown for main effects (PPFD during mother plant cultivation and propagation) and the interaction (PPPFD-M * PPFD-R) between PPFD during mother plant cultivation (PPPFD-M) and PPFD during propagation (PPPFD-R). The legend and P values are representative of both panels in the same row.
Fig. 7.
Effects of photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation and propagation on cutting dry mass and leaf area of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Cutting dry mass (g per cutting); (C, D) leaf area; (E, F) and specific leaf area. Data points indicate means of three blocks each consisting of nine replicate plants. Error bars represent the standard error of means derived from the analysis of variance. Data has been pooled for mother plant age 65 and 156 DAP. P values, when significant, are shown for main effects (PPFD during mother plant cultivation and propagation) and the interaction (PPPFD-M * PPFD-R) between PPFD during mother plant cultivation (PPPFD-M) and PPFD during propagation (PPPFD-R). The legend and P values are representative of both panels in the same row.
Mother Plant Age and Light Intensity Minimally Alter Adventitious Root Formation in Medicinal Cannabis
Authors:
,
,
,
,
, and
Click on author name to view affiliation information
Apical stem cuttings are the primary method for propagation in medicinal cannabis, yet propagation has not been studied as extensively as the later stages of crop cultivation. This study examined how mother plant age and photosynthetic photon flux density (PPFD) during mother plant cultivation and propagation affect rooting, growth, and development of apical stem cuttings. Mother plants (Cannabis sativa ‘Original Blitz’ and ‘King Harmony’) were grown in climate-controlled chambers under two light intensities (400 and 800 μmol·m−2·s−1) for up to 6 months. Apical stem cuttings were excised every 3 weeks and subsequently propagated without externally applied auxin for 3 weeks under three light intensities (50, 150, and 250 μmol·m−2·s−1). Mother plant age did not affect rooting (root dry mass and fraction of rooted cuttings). However, older mother plants exhibited decreased cutting dry mass at severance, which coincided with a reduced leaf area. The light intensity during mother plant cultivation had genotype-specific effects, with rooting either reduced or unaffected for the higher light intensity. This reduction coincided with an accumulation of starch and soluble sugar at the stem base at severance, while auxin concentrations in the apex, leaf, and stem base were unaffected by light intensity during mother plant cultivation. In contrast, light intensity during propagation did not affect the fraction of rooted cuttings. However, higher light intensity increased root and cutting dry mass. These findings indicate that mother plant age, up to 6 months, does not impact rooting in stem cuttings. However, higher light intensity during mother plant cultivation reduced rooting genotype-dependently, whereas higher light intensity during propagation increased root dry mass without affecting fraction of rooted cuttings.
Medicinal cannabis (Cannabis sativa) has garnered scientific interest for its therapeutic benefits, leading to legislation for research and medicinal use in numerous countries (Andre et al. 2016; Seddon and Floodgate, 2020; Simiyu et al. 2022). This herbaceous plant produces a variety of specialized metabolites, including cannabinoids, terpenoids, and phenolic compounds, primarily in the glandular trichomes of female inflorescences (Andre et al. 2016; Booth and Bohlmann 2019; Potter and Duncombe 2012; Spitzer-Rimon et al. 2019). Clonal propagation through apical stem cuttings (referred to as stem cuttings) excised from mother plants (also referred to as stock or donor plants) is used to ensure consistent phytochemical profiles, therapeutic efficacy, and cost-efficient production (Adhikary et al. 2021; Caplan 2018; Potter 2014).
In large-scale medicinal cannabis cultivation, apical stem cuttings are obtained from genotype-verified, virus-free mother plants to ensure uniformity and plant health (Potter 2014). Most medicinal cannabis genotypes are facultative short-day plants, which enables the maintenance of mother plants for a prolonged period in a vegetative state under long-day photoperiods exceeding 14 h of light, thereby preventing flowering (Ahrens et al. 2023; Dowling et al. 2021). Mother plants are pruned to encourage the growth of numerous branches with uniform young shoot tips, which are then periodically harvested as propagation materials (Zheng 2022). Stem cuttings are prepared by removing excessive leaves and lateral buds and are inserted into rooting substrates. Typically, the stem base is dipped into a powder or gel containing indole-3-butyric acid (IBA) or 1-naphthaleneacetic acid (NAA) to increase rooting (Zheng 2022). However, the use of synthetic auxins is not allowed in organic farming in some countries, highlighting the necessity for alternative methods, such as mother plant treatments, to promote rooting (Andersen and Bertram 1992; Lenton et al. 2018; Taylor and Birkett 2020).
Rooting is an early form of reproductive growth. Earlier studies have investigated the effect of mineral nutrition (Kpai et al. 2024; Morad and Bernstein 2023; Saloner and Bernstein 2020; Shiponi and Bernstein 2021) and leaf number and cutting position (Caplan et al. 2018) on vegetative growth. Propagation by stem cuttings involves adventitious root formation, a process influenced by a range of environmental and internal factors; specifically, in stem cuttings, it is initiated by the wounding response and changes in phytohormone concentrations within the propagation material (Druege et al. 2019; Steffens and Rasmussen 2016). The wounding response coordinates the development of a carbohydrate sink at the excision site, allowing parenchyma cells to evolve into dome-shaped adventitious root primordia. These primordia then elongate into roots, illustrating a complex interplay of hormonal regulation and cellular differentiation (Ahkami et al. 2009; Druege et al. 2019; Guan et al. 2019).
The mother plant significantly influences rooting of stem cuttings by providing auxin and carbohydrates (Osterc et al. 2009; Otiende et al. 2017). Rooting of stem cuttings is reduced with the age of the mother plants, as seen in Prosopis alba (white carob tree) (De Souza and Felker 1986), Prunus subhirtella (spring cherry) (Kunc et al. 2025), Prunus subhirtella (ornamental cherry) (Osterc et al. 2009, 2013), Pisum sativum (pea) (Rasmussen et al. 2015), and Chrysanthemum (de Ruiter 1993). Reduced rooting has been attributed to reduction in leaf area in Chrysanthemum (de Ruiter 1993), reduction in indole-3-acetic acid (IAA) (Osterc et al. 2009) or soluble sugar at the stem base with maturing tissue in ornamental cherry (Osterc et al. 2013), or reduced auxin sensitivity in Tectona grandis (teak) (Husen and Pal 2007) and pea (Rasmussen et al. 2015). Reductions in rooting associated with aging have been observed in various woody species (Diaz-Sala 2014; Mitchell et al. 2004). However, to the best of our knowledge, the impact of mother plant age on rooting of stem cuttings of medicinal cannabis has not yet been investigated.
Rooting requires the accumulation of carbohydrates at the root regeneration zone following severance from the mother plant. The translocation of carbohydrates from the leaves to the stem is stimulated by endogenous auxin levels, emphasizing the interconnected roles of carbohydrate allocation and auxin accumulation at the stem base in supporting root formation (Altman and Wareing 1975; Haissig 1986; Mishra et al. 2009; Veierskov et al. 1982). Carbohydrate accumulation in Rosa (rose) stem cuttings has conflicting effects on rooting, where rooting is either increased by starch accumulation (Carlson 1929) or there is no effect on rooting (Brandon 1940). The relationships governing carbohydrate dynamics during propagation can be categorized into four aspects (Costa et al. 2017): 1) existing carbohydrate reserves at the time of stem cutting severance; 2) carbohydrates synthesized through leaf photosynthesis during propagation; 3) reduced carbohydrate availability due to high respiration rates or the growth of non-root organs; and 4) partitioning of carbohydrates between preexisting sinks (stem and leaf) and newly developing sinks (roots) (Costa and Challa 2002; Costa et al. 2017). Regarding point 1), mother plant age and growing conditions, particularly the light intensity, can significantly affect rooting through carbohydrate accumulation (Eliasson and Brunes 1980; Hansen et al. 1978; Hansen and Eriksen 1974; Veierskov et al. 1982) or morphological differences of the stem cutting (Leakey and Storeton-West 1992). Higher light intensity increased carbohydrate concentrations at the stem base of Pinus banksiana (jack pine) (Haissig 1990) and Pelargonium (Rapaka et al. 2005) cuttings, coinciding with increased rooting (Haissig 1990). Similar relationships between carbohydrate concentrations and rooting have been reported in Eucalyptus (Hoad and Leakey 1996), Chrysanthemum (Druege et al. 2000), and Petunia (Klopotek et al. 2010). It has been proposed that assimilate supply primarily influences root growth (Noland et al. 1997), whereas auxins more strongly regulate root initiation (Ahkami et al. 2013; Blakesley 1994; Goldfarb et al. 1998), as reflected by a peak increase in endogenous auxin at the stem base within hours of severance (Agulló‐Antón et al. 2014).
However, higher light intensity during propagation inhibits or reduces root formation in various plant species due to its antagonistic interaction with auxin (Galston 1948; Gorter 1965; Nanda et al. 1968; Pierik and Steegmans, 1975; Torrey 1952, 1958). High light intensity reduced rooting of Phaseolus mungo (mung bean) cuttings that were grown without exogenous applied auxin, possibly explained by the metabolic breakdown of endogenous auxin (Jarvis and Shaheed 1987). Furthermore, higher light intensity (38 W·m−2 compared with 16 W·m−2) during mother plant cultivation in pea decreased rooting but increased auxin transport and accumulation at the stem base of stem cuttings. This effect was proposed to result from increased plant temperature, which was 27 °C under 16 W·m−2 and 33 °C under 38 W·m−2 (Baadsmand and Andersen 1984; Veierskov et al. 1982). In Chrysanthemum, higher light intensities during mother plant cultivation either decreased or increased rooting and auxin content of stem cuttings (Fischer and Hansen 1977; Tompsett and Schwabe 1974; Weigel et al. 1984). More recently, and in contradiction with previous studies, it was shown that higher light intensity (566 compared with 63 µmol m−2·s−1) increased endogenous auxin concentrations in Glycine max (soybean) (Yang et al. 2018). Currently, it is unknown if light intensity during mother plant cultivation affects auxin and carbohydrate concentrations in medicinal cannabis and if it has a subsequent effect on rooting stem cuttings. The mother plant’s response to light intensity is diverse and may depend on geographical origin. Medicinal cannabis is known for its high photosynthetic capacity (Holweg et al. 2024, 2025; Sae-Tang et al. 2024), possibly reflecting its lower-latitude origins (Zhang et al. 2018).
In addition to the effect of light intensity during mother plant cultivation, light intensity during propagation also led to different rooting responses in different plant species (Christiaens et al. 2016; Jo et al. 2008; Kurilčik et al. 2008). Increasing light intensity during propagation from 13 µmol·m−2·s−1 to 27 µmol·m−2·s−1 in Achillea millefolium (common yarrow) increased fraction of rooted cuttings, root biomass, length, and number. However, further increases to 69 µmol·m−2·s−1 led to a reduction in both fraction of rooted cuttings and root biomass (Araújo et al. 2021). Higher light intensity during propagation could also negatively impact rooting of stem cuttings by increasing transpiration, which may lead to desiccation (Tombesi et al. 2015).
The aim of this study was to investigate the effects of mother plant age and light intensity during both mother plant cultivation and propagation on rooting and the morphology of apical stem cuttings in medicinal cannabis. In addition, we examine whether carbohydrates and auxin mediate these effects. We hypothesize that stem cuttings from older mother plants show reduced rooting due to a decline in endogenous auxin concentrations. Furthermore, we hypothesize that higher light intensity during mother plant cultivation reduces rooting by decreasing auxin concentrations, whereas higher light intensity during propagation increases both plant and root dry mass.
Material and Methods
Two experiments were conducted, each using two medicinal cannabis genotypes in climate-controlled chambers. The first experiment investigated the effects of mother plant age and light intensity during mother plant cultivation on stem cutting morphology, rooting, and endogenous auxin and carbohydrate concentrations. The second experiment explored how light intensity during propagation affected stem cutting morphology and rooting.
Plant material and mother plant growth conditions.
Cannabis sativa plants {‘Original Blitz’ [Chemotype I, Delta-9-Tetrahydrocannabinol (THC) dominant] and ‘King Harmony’ [Chemotype II, THC: Cannabidiol (CBD) balanced]; Perfect Plants, Honselersdijk, the Netherlands} were cultivated in a climate-controlled chamber. This chamber was divided into eight sections using white plastic sheets. ‘Original Blitz’ and ‘King Harmony’ were selected as these are known to root either quickly [within 14 d after severance (DAS), ‘Original Blitz’] or slowly (within 21 DAS, ‘King Harmony’). Thirty-two genetically uniform mother plants from each genotype were obtained through tissue culture. The chronological age of these mother plants was recorded on a weekly basis, commencing from week 1 [0 d after planting (DAP)], when the tissue-cultured plantlets, ∼5 cm in height, arrived in the climate-controlled chamber.
Plants were transplanted into 2-L pots on 7 DAP and 5-L pots on 28 DAP, using a soil and coco substrate (Jongkind WAG11 coco substrate; Jongkind B.V., Aalsmeer, the Netherlands). The final transplant, on 42 DAP, was done into 12.5-L Air-Pots (Caledonian Tree Co. Ltd, Prestonpans, UK) using a perlite and coco substrate (Substraatmix Coco Perlite 70/30; Gold Label, Aalsmeer, the Netherlands). Plant density was 32 plants/m2 from 0 DAP (2-L pots), 16 plants/m2 from 21 DAP (2-L pots), eight plants/m2 from 28 DAP (5-L pots), and four plants/m2 from 42 DAP (12.5-L Air-Pots). On 35 DAP, to encourage the development of new shoots from a uniform canopy level, the apical meristem and the two lowest primary branches were removed. From 1 DAP onward, environmental conditions were as follows: 16-h photoperiod, air temperatures of 22/20 °C (day/night), 75% relative humidity (RH), and CO2 of 800/400 µmol·mol−1 (day/night). From 7 DAP onward, air temperature was set to 28/24 °C, and RH was reduced to 70%. The nutrient solution had an electrical conductivity (EC) of 2.2 dS·m−1 and a pH of ∼5.5 (Supplemental Table 1). The nutrient solution was applied by drip irrigation four to six times daily, at a rate of 60 mL·min−1, and a duration between 2 and 4 minutes, depending on the irrigation demand for healthy plant growth.
Mother plant light treatments and stem cutting harvest.
PPFD at canopy height was either 400 or 800 µmol m−2·s−1 provided by white LEDs (Dutch Lighting Innovations; LED TopLight 800 FS-DC, DLI APEX-Series, Aalsmeer, the Netherlands; with a spectrum of 12 Blue:32 Green:56 Red). The PPFD at canopy height was 80 µmol·m−2·s−1 on 1 DAP, increased to 250 µmol·m−2·s−1 on 7 DAP, and further increased to 400 µmol·m−2·s−1 at 14 DAP. Subsequently, on half of the plots, PPFD was increased to 800 µmol·m−2·s−1 on 42 DAP. Weekly PPFD (400–700 nm) measurements using a quantum sensor (MQ-610; Apogee Instruments Inc., Logan, CA, USA) were conducted across nine points per plot, and the dimming of the lighting fixtures was adjusted accordingly to ensure uniform PPFD conditions within a plot at canopy height. Stem cutting harvest and propagation experiments started on 65 DAP, following a 23-d acclimation period to treatment light intensity. These experiments were conducted every 3 weeks, resulting in six harvest moments on 65, 88, 112, 134, 156, and 178 DAP (of which 112 DAP was removed due to overwatering of the stem cuttings), each harvest moment representing a different mother plant age. Four mother plants per plot provided 74 unrooted stem cuttings, measuring 10 cm in length and possessing one fully expanded leaf with removed axillary buds. Of these, 54 stem cuttings were allocated for propagation experiments, whereas the remaining 20 were used for destructive measurements. Each harvest reduced the canopy height of the mother plants by 20 cm (Fig. 1).
Fig. 1.Photographs of Cannabis sativa mother plants just after [A, C; 112 days after planting (DAP)] or just before severance (B, D; 133 DAP) as well as a photograph of an apical stem cutting (E), for genotype ‘Original Blitz’ (A, B) and genotype ‘King Harmony’ (C, D, E).
Stem cuttings were allocated across 48-cell nursery trays (25 cm × 50 cm × 4 cm) equipped with transparent covers and ventilation slides. These trays were filled with vermiculite (Agra-Vermiculite Type III; Agrifield, Ommen, the Netherlands). The substrate was then soaked by submersion in a nutrient solution with an EC of 1.5 dS·m−1 and a pH of ∼5.8 for 5 min to prepare the cuttings for propagation. Details of the ion composition can be found in Supplemental Table 1. To maintain optimal humidity levels and minimize water loss through transpiration, the ventilation slides on the nursery trays were kept closed until 6 DAS from the mother plant. The transparent cover was removed from the nursery trays at 14 DAS. Propagation experiments took place within a climate-controlled chamber with three tiers, where each tier was dedicated to a light treatment and accommodated three nursery trays. Environmental conditions within the climate chamber were a 16 h photoperiod, air temperatures of 22/20 °C (day/night), 75% RH, and CO2 of 450 µmol·mol−1.
Propagation light treatments.
Six stem cutting harvests were conducted to assess the effects of mother plant age and light intensity during mother plant cultivation (65, 88, 112, 134, 156, and 178 DAP; 112 DAP was removed from analysis due to overwatering). Each harvest included a control treatment with a PPFD of 150 µmol·m−2·s−1 during propagation to evaluate the effect of mother plant age on rooting. In addition, two of the six harvests were used for light intensity propagation experiments, that is, the first (65 DAP) and fifth (156 DAP) harvests. These experiments investigated the effect of light intensity on rooting, with canopy-level PPFD set at 50, 150, or 250 µmol·m−2·s−1 (GreenPower LED Low Blue. Generation 2; Philips, Eindhoven, the Netherlands; with a spectrum of 13 Blue:17 Green:70 Red).
Destructive measurements.
Destructive morphological measurements were conducted on 0, 14, and 21 DAS. For the propagation trials, nine stem cuttings (three per nursery tray) from each mother plant and propagation PPFD plot were harvested for intermediate (14 DAS) and final (21 DAS) harvest, resulting in a total of 216 cuttings for each harvesting moment. Measurements included leaf area and fresh and dry mass of the stem and leaves. In addition, for the intermediate and final harvest, measurements included fraction of rooted cuttings (fraction of stem cuttings with at least one root exceeding 5 mm) and root dry mass. Dry mass data were determined for each set of three stem cuttings per nursery tray, whereas other variables were measured per individual cutting and averaged. Dry mass was determined using a ventilated oven (24 h at 70 °C, followed by 48 h at 105 °C). Leaf area was determined using an LI-3100C area meter (LI-COR Inc., Lincoln, NE, USA).
Auxin and carbohydrate analysis.
Apex, the youngest fully expanded leaf and stem base of stem cuttings excised on 65, 134, and 178 DAP were taken between 2 and 4 hours after the light fixtures were turned on for auxin and carbohydrate analysis. The samples were immediately preserved in liquid nitrogen at severance and stored at −80 °C. A ball mill ground the frozen organ samples at 80 Hz for 20 s. Auxin extraction was performed as previously described by Ruyter-Spira et al. (2011). A 0.01-g fresh weight powder of the apex or stem base was extracted with 1 mL of ice-cold methanol (MeOH) containing (phenyl 13C6)-IAA (0.1 nmol·mL−1) as an internal standard in a 2-mL Eppendorf tube. The tubes were vortexed and sonicated for 10 min in an ultrasonic water bath (Branson 3510; Branson Ultrasonics, Eemnes, The Netherlands) and placed overnight in an orbital shaker at 4 °C. Next, samples were centrifuged for 10 min at 11,500 rpm in a centrifuge (Heraeus Fresco 17; Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C, after which the organic phase was loaded on a 100-mg 1.5-mL Extra-Clean SPE Amino cartridge (S*Pure Pte. Ltd., Singapore). The cartridge was equilibrated before sample loading and subsequently was washed and eluted. The MeOH was evaporated in a speed vacuum system (SPD121P, Thermo Savant, Hastings, UK) at room temperature and the residue resuspended in 100 μL acetonitrile:water:formic acid (20:80:0.1, vol/vol/vol). The samples were filtered through a 0.45-μm filter (Minisart SRP4, Sartorius, Goettingen, Germany) and measured on the same day. IAA was analyzed using a Waters Xevo TQ tandem quadruple mass spectrometer (Sae-Tang et al. 2024).
Carbohydrate extraction and analysis were conducted according to Sae-Tang et al. (2024), and the frozen ground organs were freeze-dried. A 15-mg dry weight powder of each of the three plant organs was extracted with 5 mL of 80% ethanol at 80 °C for 20 min in a shaking water bath. Then, the extracts were centrifuged for 5 min at 8500 relative centrifugal force (rcf) at 4 °C (Universal 320R; Hettich, Geldermalsen, the Netherlands). One milliliter of supernatant was transferred to a 2-mL Eppendorf tube and dried in a vacuum centrifuge (Savant SpeedVac SPD2010; Thermo Fisher Scientific) at a setting of 50 °C and 5.1 Torr for 120 min. The pellet with the remaining supernatant was stored for starch measurement at −20 °C. The dried samples in the Eppendorf tube were resuspended in 1 mL Mili-Q water and sonicated in an ultrasonic water bath (Branson 2800; Branson Ultrasonics) for 10 min at 4 °C. The solutions were centrifuged at 21,100 rcf for 10 min (Sorvall Legend Micro 21R; Thermo Fisher Scientific). After 10 times dilution with Milli-Q water, glucose, fructose, and sucrose were quantified using a High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD; Dionex ICS5000, Thermo Fisher Scientific) equipped with a CarboPac PA1 column (250 × 2 mm) (Thermo Fisher Scientific) eluted with 100 mM NaOH at a flow rate of 0.25 mL·min−1 at 25 °C. Chromeleon 7.2 (Thermo Fisher Scientific) was used for data analysis. Total soluble sugar was calculated as the sum of glucose, fructose, and sucrose. The stored pellet was used for starch analysis. After washing three times with 80% ethanol, the pellet was dried in a vacuum centrifuge (Savant SpeedVac SPD2010; Thermo Fisher Scientific) at 55 °C and 5.1 Torr for 25 min then resuspended in 2 mL of 1 g·L−1 thermostable alpha-amylase (SERVA Electrophoresis GmbH) in Milli-Q water and incubated for 30 min at 90 °C in a shaking water bath. Then 1 mL of 0.5 g·L−1 amyloglucosidase (Sigma 10115) in 50 mM citrate buffer (pH 4.6) was added and incubated at 60 °C for 10 min in a shaking water bath. After centrifugation at 21,100 rcf for 10 min and 20 to 50 times dilution with Milli-Q water, glucose was quantified using HPEAC-PAD as described above. Starch content was determined by measuring the amount of glucose released from the hydrolysis of soluble material and multiplying it by a conversion factor of 0.9, which accounts for the fact that during starch hydrolysis, a cleaved starch unit (molecular weight 162) combines with a water molecule (molecular weight 18) to form a glucose molecule, with the ratio 162/(162 + 18) = 0.9.
Statistical analysis.
This study consisted of two experiments. The mother plant experiment was set up and analyzed as a split-split-plot design in two blocks (within the climate-controlled chamber) with light intensity during mother plant cultivation (400 and 800 µmol·m−2·s−1) as the main factor, genotype (‘Original Blitz’ and ‘King Harmony’) as subfactor, and mother plant age (65, 88, 134, 156, and 178 DAP) as sub-subfactor. Each block consisted of four replicate plants per treatment plot, which provided unrooted stem cuttings for the propagation experiments. The propagation experiment was set up and analyzed as a split-split-plot design in two blocks (mother plant ages 65 and 156 DAP) with light intensity during propagation (50, 150, or 250 µmol·m−2·s−1) as the main factor and light intensity during mother plant cultivation (400 and 800 µmol·m−2·s−1) as subfactor, and genotype (‘Original Blitz’ and ‘King Harmony’) as sub-subfactor. Each block in the propagation experiment consisted of 18 stem cuttings dispersed over three nursery trays, of which nine replicate cuttings were harvested at an intermediate harvest (14 DAS) and nine replicate cuttings at the final harvest (21 DAS). For auxin and carbohydrate measurements in the mother plant experiment, the cuttings were derived from three different mother plant ages (65, 134, and 178 DAP). Individual plant responses were averaged per plot and an average was used as a statistical replicate. No outliers were identified per plot, using Z-score criteria, with thresholds set at −3 and +3 standard deviations. Due to the limited number of blocks, homogeneity of variances could not be tested and, therefore, was assumed. A Shapiro-Wilk test ascertained that the assumption of normality was met for all variables tested. Analysis of variance was performed with mean separation by protected least significant difference test. To minimize the risk of overlooking potential treatment effects, given the low number of blocks in the mother plant and propagation experiment, a threshold of P = 0.10 was used (Holweg et al. 2024, 2025; Ott and Longnecker 2015). Statistical analysis was conducted using GenStat (21st edition, VSN International LTD, Hemel Hempstead, UK).
Results
Plant age and light intensity during mother plant cultivation.
Surprisingly, in both genotypes mother plant aging from 65 to 178 DAP did not reduce fraction of rooted cuttings and root dry mass of stem cuttings at 14 or 21 DAS (Fig. 2A–D and Supplemental Fig. 1A–D). In both genotypes, fraction of rooted cuttings at 14 DAS even increased as mother plants aged (Supplemental Fig. 1A–B), with no apparent effect on root dry mass (Supplemental Fig. 1C–D). However, stem cuttings harvested from older mother plants in both genotypes had a lower dry mass and leaf area (Fig. 3A–D). This further translated into lower cutting dry mass in both genotypes at 14 DAS. However, no effect was observed at 21 DAS, indicating that growth during propagation compensated for the smaller size at severance for cuttings from older mother plants (Supplemental Fig. 2C–F). Light intensity during mother plant cultivation showed genotype-specific responses where the fraction of rooted cuttings and root dry mass of stem cuttings harvested from the quick rooting ‘Original Blitz’ were unaffected at 14 or 21 DAS (Fig. 2A and 2C and Supplemental Fig. 1A and 1C). Higher light intensity during mother plant cultivation reduced fraction of rooted cuttings and root dry mass of stem cuttings harvested from the slow-rooting ‘King Harmony’ at both 14 and 21 DAS (Fig. 2B and 2D and Supplemental Fig. 1B and 1D). Higher light intensity during mother plant cultivation increased cutting dry mass in both genotypes at 0 (Fig. 3A–B) and 14 DAS (Supplemental Fig. 2C and 2D) but only resulted in increased cutting dry mass at 21 DAS in ‘Original Blitz’ (Supplemental Fig. 2E and 2F). Furthermore, higher light intensity during mother plant cultivation did not affect leaf area at 0 DAS (Fig. 3C and 3D) but decreased specific leaf area (SLA) in both genotypes (Supplemental Fig. 2A and 2B).
Fig. 2.Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on rooting of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Fraction of rooted cuttings (C, D) and root dry mass (g per cutting) of rooted cuttings. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 3.Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on cutting dry mass and leaf area of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Cutting dry mass (g per cutting) (C, D) and leaf area. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Auxin and carbohydrate content in stem cuttings at severance.
The concentrations of IAA in the apex, leaf, and stem base of cuttings at severance differed among mother plant ages, IAA concentrations declined around the midpoint of mother plant cultivation (134 DAP) and later increased at 175 DAP (Fig. 4). This trend was observed in both genotypes and was more pronounced in the stem base IAA concentrations (Fig. 4E and 4F). The light intensity during mother plant cultivation did not influence IAA concentrations in any of the three cutting organs for either genotype (Fig. 4). Starch content in the leaf was higher in cuttings from mother plants grown at 800 compared with 400 μmol·m−2·s−1, with a tendency to increase starch in the apex and stem base (Fig. 5A–F). Cuttings of older mother plants showed reduced starch content in all tissues at severance (Fig. 5A–F). Higher light intensity during mother plant cultivation increased total soluble sugar content in the apex of both genotypes (Supplemental Fig. 3E and 3F). At the same time, effects in the leaf and stem were variable (Supplemental Fig. 3). Furthermore, increasing mother plant age reduced total soluble sugar in the stem base, with no effect in the apex and leaf (Supplemental Fig. 3).
Fig. 4.Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on indole-3-acetic acid (IAA) concentration in organs of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) in apex; (C, D) in leaf; (E, F) and in stem. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 5.Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on starch content in organs of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) In apex; (C, D) in leaf; (E, F) and in stem. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
No interaction was observed between the light intensity during mother plant cultivation and light intensity during propagation. Higher light intensity during propagation did not affect fraction of rooted cuttings at 14 and 21 DAS in either genotype (Fig. 6A and B and Supplemental Fig. 4A and 4B). However, higher light intensity during propagation increased root and cutting dry mass at 14 DAS in both genotypes (Supplemental Figs. 4C and 4D and 5A and 5B), with no effect at 21 DAS (Figs. 6C and 6D and 7A and 7B). Higher light intensity during propagation did not affect leaf area at 14 (Supplemental Fig. 5C and 5D) and 21 DAS (Fig. 7C and 7D) in either genotype, although SLA was decreased at 14 DAS in both genotypes (Supplemental Fig. 5E and 5F) and unaffected by light intensity at 21 DAS (Fig. 7E and 7F).
Fig. 6.Effects of photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation and propagation on rooting of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Fraction of rooted cuttings (C, D) and root dry mass (g per cutting) of rooted cuttings. Data points indicate means of three blocks each consisting of nine replicate plants. Error bars represent the standard error of means derived from the analysis of variance. Data have been pooled for mother plant age 65 and 156 DAP. P values, when significant, are shown for main effects (PPFD during mother plant cultivation and propagation) and the interaction (PPPFD-M * PPFD-R) between PPFD during mother plant cultivation (PPPFD-M) and PPFD during propagation (PPPFD-R). The legend and P values are representative of both panels in the same row.
Fig. 7.Effects of photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation and propagation on cutting dry mass and leaf area of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Cutting dry mass (g per cutting); (C, D) leaf area; (E, F) and specific leaf area. Data points indicate means of three blocks each consisting of nine replicate plants. Error bars represent the standard error of means derived from the analysis of variance. Data has been pooled for mother plant age 65 and 156 DAP. P values, when significant, are shown for main effects (PPFD during mother plant cultivation and propagation) and the interaction (PPPFD-M * PPFD-R) between PPFD during mother plant cultivation (PPPFD-M) and PPFD during propagation (PPPFD-R). The legend and P values are representative of both panels in the same row.
This study explored the effects of mother plant age and light intensity during cultivation and propagation on rooting, cutting dry mass, and stem cutting morphology in medicinal cannabis.
Mother plant age did not affect rooting but decreased cutting dry mass and leaf area at severance.
Mother plant age did not affect rooting (Fig. 2A–D and Supplemental Fig. 1A–D). This contradicts studies in which stem cuttings from older mother plants have reduced capability of cells to initiate rooting and translocate carbohydrates to the root regeneration zone for root growth (Rasmussen and Hunt 2010; Tombesi et al. 2015). In addition, stem cuttings from older mother plants exhibit lower auxin concentrations (Osterc et al. 2009), a trend not observed in our study (Fig. 4). Auxin concentrations in the mother plant did not correlate to the ability to root in various genotypes of Chrysanthemum (Stoltz 1968), Acer saccharum (Greenwood et al. 1976), Dahlia (Biran and Halevy 1973), and Rhododendron (Wu and Barnes 1981). In various species, the rooting of stem cuttings decreased with increasing mother plant age (de Ruiter 1993; De Souza and Felker 1986; Kunc et al. 2025; Osterc et al. 2009, 2013; Rasmussen et al. 2015). The absence of this effect in our study could be attributed to several factors: 1) mother plant age may have no significant impact on rooting of medicinal cannabis, 2) ‘Original Blitz’ and ‘King Harmony’ are not as susceptible to reduced rooting due to mother plant age, 3) the effect of mother plant age on rooting could not be observed within 178 DAP but might be observed after prolonged periods, and 4) the reduction in rooting seen with increasing mother plant age may instead result from other factors, such as pest and disease pressure buildup due to inadequate hygiene protocols when excising stem cuttings from mother plants (Faust et al. 2016). In our study, increased mother plant age reduced total soluble sugar content in the stems of both genotypes, with no effect on sugar content in the apex or leaf (Supplemental Fig. 3). Despite this reduction, there was no effect of mother plant age on rooting of both genotypes (Fig. 2A–D). These findings align with the findings in white carob tree (De Souza and Felker 1986) and Chrysanthemum (Druege et al. 2000), where no correlation between stem carbohydrate concentration and rooting was found. However, increased carbohydrates in stem cuttings of Pelargonium increased root number, but only below 100 µmol·m−2·s−1 (Rapaka et al. 2005).
Older mother plants produced stem cuttings with lower cutting dry mass 0 and 14 DAS (Fig. 3A and 3B and Supplemental Fig. 2C and 2D) in both genotypes, of which the former coincided with a decreased leaf area (Fig. 2A–D). However, no apparent effect of mother plant age was observed on cutting dry mass 21 DAS (Supplemental Fig. 2E and 2F). Similarly, increasing mother plant age decreased the fresh mass of excised stem cuttings in Chrysanthemum (Agustsson and Canham 1982; Anderson and Carpenter 1974; Eng et al. 1985) and reduced leaf area in excised teak cuttings (Husen and Pal 2007). The reduced cutting dry mass at 14 DAS in our study (Supplemental Fig. 2C and 2D) likely resulted from lower light interception due to decreased leaf area at 0 DAS (Fig. 3C and 3D), which limited photon capture and plant growth. However, because medicinal cannabis roots rapidly, the substrate may have constrained root and plant growth at some moment, explaining the absence of statistical differences in cutting dry mass at 21 DAS (Semchenko et al. 2007). In stem cuttings of roses, root dry mass linearly correlated with the leaf area of these cuttings at severance (Costa and Challa 2002). Leaves are important in shoot and root development as these provide and store carbohydrates and auxin (Davis and Haissig 1994; Sandhya et al. 2022; Tombesi et al. 2015).
Higher light intensity during mother plant cultivation reduced rooting genotype-dependently, independent of auxin and carbohydrates.
Higher light intensities during mother plant cultivation reduced rooting of one of the two genotypes (Fig. 2A–D and Supplemental Fig. 1A–D). The positive effect of lower light intensities during mother plant cultivation on the rooting ability of excised stem cuttings has been observed in multiple plant species (Eliasson and Brunes 1980; Hansen et al. 1978; Hansen and Eriksen 1974; Leakey and Storeton-West 1992). It has been proposed that higher light intensities during mother plant cultivation in Chrysanthemum could decrease endogenous auxin concentrations, subsequently reducing rooting (Weigel et al. 1984), although contradicting effects have also been reported (Fischer and Hansen 1977). In our study, mother plant age did not affect auxin concentrations nor rooting (Fig. 4). Furthermore, Costa and Challa (2002) confirmed that auxins do not increase rooting of roses when leaves are absent or covered, suggesting that photosynthesis and photosynthates play a primary role in regulating rooting rather than the leaves’ ability to supply auxin. In addition to carbohydrates and auxins, rhizocaline and other unidentified auxin synergists have been proposed as rooting cofactors, which are hypothetical root-promoting substances that are translocated from leaves to the stem base (Wilson and Staden 1990). Initial carbohydrate levels in stem cuttings did not correlate with increased fraction of rooted cuttings (Hansen et al. 1978; Veierskov 1988; Veierskov et al. 1982), and the effectiveness of carbohydrate reserves in promoting rooting also depends on the light intensity during propagation (Rapaka et al. 2005). In our study, mother plant cultivation at a higher light intensity increased starch accumulation in the leaves and tended to increase in the apex and stem base (Fig. 5). A similar response was observed in Pelargonium mother plants grown at a higher light intensity, where photosynthates accumulated in the stem base (Rapaka et al. 2005), and other plant organs of the excised cutting equally (Davis and Potter 1981; Veierskov et al. 1982).
The increase in total soluble sugar content in the stem of ‘Original Blitz’ with higher light intensity (Supplemental Fig. 3E) did coincide with an increased fraction of rooted cuttings and root dry mass (Fig. 2A and 2C and Supplemental Fig. 1A and 2C). On the contrary, in ‘King Harmony’ the increase in total soluble sugar in the stem with a higher light intensity (Supplemental Fig. 3F) coincided with a decrease in rooting (Fig. 2B and 2D and Supplemental Fig. 1B). This is consistent with the findings of Hansen and Eriksen (1974), who observed a decrease in root number in pea cuttings when mother plants were grown under a higher light intensity. The authors proposed that this was due to suboptimal carbohydrate content in relation to auxin content. Furthermore, Veierskov et al. (1982) found that carbohydrate content increased in pea mother plants when grown at a higher light intensity. In contrast, no subsequent effect on rooting of excised stem cuttings was found. Increased carbohydrate accumulation could negatively affect photosynthesis due to feedback inhibition and possibly affect rooting (Hansen et al. 1978; Iglesias et al. 2002; Leakey and Storeton-West 1992). Higher light intensity during mother plant cultivation increased soluble sugar content in the stems, whereas sugar levels in the apex and leaf tended to increase (Supplemental Fig. 3B, 3D, and 3F). This accumulation might have reduced rooting of ‘King Harmony,’ potentially causing end-product inhibition of photosynthesis (Leakey and Storeton-West 1992). Similarly, Agulló-Antón et al. (2011) showed that Dianthus caryophyllus (carnation) stem cuttings stored in the light rather than in darkness accumulated supra-optimal soluble sugar concentrations in the stem base, thereby inhibiting growth, a phenomenon first described by Hansen and Eriksen (1974).
An interactive effect between light intensity during mother plant cultivation, mother plant age, and genotype was observed across multiple parameters in this study. For the slow-rooting genotype ‘King Harmony’, the accumulation of carbohydrates and auxins under higher light intensities may have led to negative feedback inhibition of photosynthesis, thereby reducing rooting capacity. Alternatively, ‘King Harmony’ may possess a lower tolerance to higher concentrations of these compounds, resulting in suboptimal conditions for rooting. These findings highlight the importance of considering genotype-specific responses to light intensity on rooting.
Higher light intensity during propagation increased root growth without affecting fraction of rooted cuttings.
Higher light intensity during propagation did not affect the fraction of rooted cuttings and increased root dry mass at 14 DAS (Supplemental Fig. 4A–D), which could potentially increase plant growth in subsequent growth phases, as seen in Chrysanthemum (Hicklenton 1984). The increase in root dry mass with a higher light intensity agrees with previous studies in which the production of photo-assimilates was positively correlated with root dry mass (Costa and Challa 2002; Tombesi et al. 2015). After cutting severance from the mother plant, leaf photosynthesis rate drops significantly due to the imbalance between water loss and water supply and subsequent closure of stomata (Fordham et al. 2001; Smalley et al. 1991; Svenson et al. 1995). The decrease in leaf photosynthesis continues until rooting restores the water balance of the cutting (Eliasson and Brunes 1980; Gay and Loach 1977). Maintaining a higher light intensity during propagation before the formation of roots would be counterintuitive, as CO2 diffusion into the leaf would be limited due to the closure of stomata (Momayyezi et al. 2022). In addition, a higher light intensity during propagation can increase plant temperature and enhance water stress, which potentially has a negative effect on rooting (Tombesi et al. 2015). However, water stress was not observed in this study. The photosynthetic activity and plant temperature of the stem cutting was not measured, although there was no adverse effect of higher light intensity during propagation on a fraction of rooted cuttings or root dry mass.
It should be noted that no exogenous auxin was applied in this experiment. Specifically, no rooting powders or gels containing IBA or NAA, both known to promote rooting (Zheng 2022). Given the relatively high rooting rates observed throughout our study, it can be proposed that the effects of the light treatments on rooting might become less evident if a rooting agent were applied, as this could potentially mask treatment-specific differences.
Conclusion
Mother plant age (up to 6 months) did not influence rooting of excised stem cuttings in medicinal cannabis, although it reduced cutting dry mass at 0 DAS, which coincided with a reduced leaf area. Higher light intensity during mother plant cultivation increased cutting dry mass in both genotypes at 0 and 14 DAS but reduced rooting of one of the two genotypes. This reduced rooting was accompanied by an increased accumulation of soluble sugar and starch at the stem base, whereas starch in the apex and leaf and auxin concentrations remained unaffected. In addition, a higher light intensity during propagation did not influence the fraction of rooted cuttings but did increase root and cutting dry mass at 14 DAS. These findings suggest that cultivating mother plants under a higher light intensity causes inhibitory effects on rooting of excised stem cuttings, although this effect appears to be genotype-dependent. Furthermore, 250 µmol·m−2·s−1 can be applied during propagation, as it did not affect a fraction of rooted cuttings compared with a lower light intensity but significantly increased root and cutting dry mass 14 DAS.
Received: 06 Jun 2025
Accepted: 02 Sept 2025
Published Online: 06 Oct 2025
Published Print: 01 Nov 2025
Fig. 1.
Photographs of Cannabis sativa mother plants just after [A, C; 112 days after planting (DAP)] or just before severance (B, D; 133 DAP) as well as a photograph of an apical stem cutting (E), for genotype ‘Original Blitz’ (A, B) and genotype ‘King Harmony’ (C, D, E).
Fig. 2.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on rooting of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Fraction of rooted cuttings (C, D) and root dry mass (g per cutting) of rooted cuttings. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 3.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on cutting dry mass and leaf area of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Cutting dry mass (g per cutting) (C, D) and leaf area. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 4.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on indole-3-acetic acid (IAA) concentration in organs of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) in apex; (C, D) in leaf; (E, F) and in stem. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 5.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on starch content in organs of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) In apex; (C, D) in leaf; (E, F) and in stem. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 6.
Effects of photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation and propagation on rooting of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Fraction of rooted cuttings (C, D) and root dry mass (g per cutting) of rooted cuttings. Data points indicate means of three blocks each consisting of nine replicate plants. Error bars represent the standard error of means derived from the analysis of variance. Data have been pooled for mother plant age 65 and 156 DAP. P values, when significant, are shown for main effects (PPFD during mother plant cultivation and propagation) and the interaction (PPPFD-M * PPFD-R) between PPFD during mother plant cultivation (PPPFD-M) and PPFD during propagation (PPPFD-R). The legend and P values are representative of both panels in the same row.
Fig. 7.
Effects of photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation and propagation on cutting dry mass and leaf area of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Cutting dry mass (g per cutting); (C, D) leaf area; (E, F) and specific leaf area. Data points indicate means of three blocks each consisting of nine replicate plants. Error bars represent the standard error of means derived from the analysis of variance. Data has been pooled for mother plant age 65 and 156 DAP. P values, when significant, are shown for main effects (PPFD during mother plant cultivation and propagation) and the interaction (PPPFD-M * PPFD-R) between PPFD during mother plant cultivation (PPPFD-M) and PPFD during propagation (PPPFD-R). The legend and P values are representative of both panels in the same row.
AdhikaryD
,
KulkarniM
,
El-MezawyA
,
MobiniS
,
ElhitiM
,
GjuricR
,
RayA
,
PolowickP
,
SlaskiJJ
,
JonesMP
,
BhowmikP
. 2021. Medical cannabis and industrial hemp tissue culture: Present status and future potential. Front Plant Sci. 12:627240.
. 2009. Molecular physiology of adventitious root formation in Petunia hybrida cuttings: Involvement of wound response and primary metabolism. New Phytol. 181(
. 2013. Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation. Planta. 238(
. 1975. The effect of IAA on sugar accumulation and basipetal transport of 14C‐labelled assimilates in relation to root formation in Phaseolus vulgaris cuttings. Physiol Plant. 33(
. 2021. Photon flux density and wavelength influence on growth, photosynthetic pigments and volatile organic compound accumulation in Aeollanthus suaveolens (Catinga-de-mulata) under in vitro conditions. Ind Crops Prod. 168:113597. https://doi.org/10.1016/j.indcrop.2021.113597.
. 2018. Vegetative propagation of cannabis by stem cuttings: Effects of leaf number, cutting position, rooting hormone, and leaf tip removal. Can J Plant Sci. 98(
. 1986. The influence of stock plant fertilisation on tissue concentrations of N, P and carbohydrates and the rooting of Prosopis alba cuttings. For Ecol Manage. 16(
. 2014. Direct reprogramming of adult somatic cells toward adventitious root formation in forest tree species: The effect of the juvenile–adult transition. Front Plant Sci. 5:310. https://doi.org/10.3389/fpls.2014.00310.
. 2000. Relation between nitrogen status carbohydrate distribution and subsequent rooting of chrysanthemum cuttings as affected by pre-harvest nitrogen supply and cold-storage. Ann Bot. 85(
. 1985. The effects of supplementary HPS lighting and carbon dioxide enrichment on the vegetative growth, nutritional status and flowering characteristics of Chrysanthemum morifolium Ramat. J Hortic Sci. 60(
. 1977. Rooting of chrysanthemum cuttings. Influence of irradiance during stock plant growth and of decapitation and disbudding of cuttings. Sci Hortic. 7(
. 2025. High air temperature reduces plant specialised metabolite yield in medical cannabis, and has genotype-specific effects on inflorescence dry matter production. Environ Exp Bot. 230:106085. https://doi.org/10.1016/j.envexpbot.2025.106085.
. 2024. The role of red and white light in optimising growth and accumulation of plant specialised metabolites at two light intensities in medical cannabis (Cannabis sativa L.). Front Plant Sci. 15:1393803.
. 2007. Metabolic changes during adventitious root primordium development in Tectona grandis Linn. f. (teak) cuttings as affected by age of donor plants and auxin (IBA and NAA) treatment. New For (Dordr). 33(
. 2010. Dark exposure of petunia cuttings strongly improves adventitious root formation and enhances carbohydrate availability during rooting in the light. J Plant Physiol. 167(
. 1992. The rooting ability of Triplochiton scleroxylon cuttings: The interactions between stockplant irradiance, light quality and nutrients. For Ecol Manage. 49(
. 2018. Growing practices and the use of potentially harmful chemical additives among a sample of small-scale cannabis growers in three countries. Drug Alcohol Depend. 192:250–256.
. 1968. Effect of sucrose, auxins and gibberellic acid on rooting of stem segments of Populus nigra under varying light conditions. Plant Cell Physiol. 9(
. 1997. The dependance of root growth potential on light level, photosynthetic rate, and root starch content in jack pine seedlings. New For (Dordr). 13(
. 2013. C
arbohydrate involvement in rooting success of leafy cuttings depending on physiological age of stock plants of Prunus subhirtella ‘Autumnalis’. Acta Hortic. 990:423–428. https://doi.org/10.17660/ActaHortic.2013.990.54.
. 2017. Effects of cutting position of rose rootstock cultivars on rooting and its relationship with mineral nutrient content and endogenous carbohydrates. Sci Hortic. 225:204–212. https://doi.org/10.1016/j.scienta.2017.07.009.
. 2014. A review of the cultivation and processing of cannabis (Cannabis sativa L.) for production of prescription medicines in the UK. Drug Test Anal. 6(
. 2005. Interplay between initial carbohydrate availability, current photosynthesis, and adventitious root formation in Pelargonium cuttings. Plant Sci. 168(
. 2011. Physiological effects of the synthetic strigolactone analog GR24 on root system architecture in Arabidopsis: Another belowground role for strigolactones?Plant Physiol. 155(
. 2024. Effect of far-red and blue light on rooting in medicinal cannabis cuttings and related changes in endogenous auxin and carbohydrates. Sci Hortic. 325:112614. https://doi.org/10.1016/j.scienta.2023. 112614.
. 2024. High light intensity improves yield of specialised metabolites in medicinal cannabis (Cannabis sativa L.), resulting from both higher inflorescence mass and concentrations of metabolites. J Appl Res Med Aromat Plants. 43:100583. https://doi.org/10.1016/j.jarmap.2024.100583.
. 2022. Adventitious root formation in cuttings as influenced by genotypes, leaf area, and types of cuttings, p 381–395. In: Environmental, physiological and chemical controls of adventitious rooting in cuttings.
. 2021. Response of medical cannabis (Cannabis sativa L.) genotypes to P supply under long photoperiod: Functional phenotyping and the ionome. Ind Crops Prod. 161:113154. https://doi.org/10.1016/j.indcrop.2020.113154.
. 2022. Understanding Cannabis sativa L.: Current status of propagation, use, legalisation, and haploid-inducer-mediated genetic engineering. Plants. 11(
. 1968. Factors influencing root initiation in an easy- and a difficult-to-root chrysanthemum. Proceedings of the American Society for Horticultural Science. 92:622–625.
. 1995. Gas exchange, water relations, and dry weight partitioning during root initiation and development of poinsettia cuttings. J Am Soc Hortic Sci. 120(
. 2015. Influence of light and shoot development stage on leaf photosynthesis and carbohydrate status during the adventitious root formation in cuttings of Corylus avellana L. Front Plant Sci. 6:973.
. 1982. Dynamics of extractable carbohydrates in Pisum sativum. I. Carbohydrate and nitrogen content in pea plants and cuttings grown at two different irradiances. Physiol Plant. 55(
. 2018. Auxin-to-gibberellin ratio as a signal for light intensity and quality in regulating soybean growth and matter partitioning. Front Plant Sci. 9:56.
We thank the following companies for their valuable input in the suppliance of material: Caledonian Tree Co. Ltd, Gold Label, Dutch Lighting Innovations, and Perfect Plants B.V. We also thank the following staff members of Wageningen University and Research for their technical support: Gerrit Stunnenberg, David Brink, Jannick Verstegen, Dieke Smit, Chris van Asselt, Sean Geurts, Martijn Verweij, and Jonathan Hovenkamp.
*
M.M.S.F.H. is the corresponding author. E-mail: mexx@dli.nl.
Photographs of Cannabis sativa mother plants just after [A, C; 112 days after planting (DAP)] or just before severance (B, D; 133 DAP) as well as a photograph of an apical stem cutting (E), for genotype ‘Original Blitz’ (A, B) and genotype ‘King Harmony’ (C, D, E).
Fig. 2.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on rooting of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Fraction of rooted cuttings (C, D) and root dry mass (g per cutting) of rooted cuttings. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 3.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on cutting dry mass and leaf area of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Cutting dry mass (g per cutting) (C, D) and leaf area. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 4.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on indole-3-acetic acid (IAA) concentration in organs of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) in apex; (C, D) in leaf; (E, F) and in stem. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 5.
Effects of mother plant age and photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation on starch content in organs of Cannabis sativa stem cuttings 0 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) In apex; (C, D) in leaf; (E, F) and in stem. Data points indicate means of two blocks each consisting of four replicate plants. Error bars represent the standard error of means derived from the analysis of variance. P values, when significant, are shown for main effects (PPFD, or mother plant age) and the interaction (PPPFD-M * Age) between PPFD during mother plant cultivation (PPPFD-M) and mother plant age (PAge). The legend and P values are representative of both panels in the same row.
Fig. 6.
Effects of photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation and propagation on rooting of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Fraction of rooted cuttings (C, D) and root dry mass (g per cutting) of rooted cuttings. Data points indicate means of three blocks each consisting of nine replicate plants. Error bars represent the standard error of means derived from the analysis of variance. Data have been pooled for mother plant age 65 and 156 DAP. P values, when significant, are shown for main effects (PPFD during mother plant cultivation and propagation) and the interaction (PPPFD-M * PPFD-R) between PPFD during mother plant cultivation (PPPFD-M) and PPFD during propagation (PPPFD-R). The legend and P values are representative of both panels in the same row.
Fig. 7.
Effects of photosynthetic photon flux density (PPFD) (400 and 800 µmol·m−2·s−1) during mother plant cultivation and propagation on cutting dry mass and leaf area of Cannabis sativa stem cuttings 21 d after severance in ‘Original Blitz’ and ‘King Harmony’. (A, B) Cutting dry mass (g per cutting); (C, D) leaf area; (E, F) and specific leaf area. Data points indicate means of three blocks each consisting of nine replicate plants. Error bars represent the standard error of means derived from the analysis of variance. Data has been pooled for mother plant age 65 and 156 DAP. P values, when significant, are shown for main effects (PPFD during mother plant cultivation and propagation) and the interaction (PPPFD-M * PPFD-R) between PPFD during mother plant cultivation (PPPFD-M) and PPFD during propagation (PPPFD-R). The legend and P values are representative of both panels in the same row.