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).

View Figure

• Download as Powerpoint
• Download Figure

Propagation growth conditions.

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).

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).

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

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).

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

Light intensity during propagation.

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).

View Figure

• Download as Powerpoint
• Download Figure

View Figure

• Download as Powerpoint
• Download Figure

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.