Plant material.

Stock plants were established from liners of day-neutral ‘Albion’ and short-day ‘Fronteras’ that were individually transplanted on 18 Nov 2022 into 20.3-cm-diameter × 14.0-cm depth containers filled with horticulture-grade substrate (Berger BM7 all-purpose mix; Berger, Saint-Modeste, QC, Canada) composed of 50% coarse peatmoss, 35% pine bark, and 15% perlite. Stock plants were placed in a glass-glazed greenhouse in West Lafayette, IN, USA (lat. 40°N) and grown in a hanging-basket system with drip irrigation. The greenhouse had retractable shade curtains, pad-and-fan evaporative cooling, and radiant hot-water-pipe heating regulated by an environmental control system (Maximizer Precision 10; Priva Computers, Vineland Station, ON, Canada). Supplemental lighting was delivered by 1000-W high-pressure sodium lamps (P.L. Light Systems Inc., Beamsville, ON, Canada) used for 16 h·d⁻¹ (0500 to 1900 HR), providing a PPFD of approximately 150 µmol·m^‒2·s^‒1 and a daily light integral (DLI) of 8.6 mol·m⁻²·d⁻¹. Air temperature and relative humidity (RH) were set at 24 °C and 70%, respectively.

The experiment was replicated three times and consisted of two phases: a propagation phase during which PPFD treatments were imposed, and a greenhouse finishing phase to evaluate carryover treatment effects. On 22 Jun, 8 Aug, and 19 Sep 2023, uniform runner tips of two different crown diameters (small <10 mm or large >10 mm), each pruned to two trifoliate leaves, were transplanted into industry-standard 42-cell propagation trays (88.7-mL individual cell volume) cut into 4 × 2 trays and filled with horticulture-grade substrate (Berger BM2 Seed Germination; Berger, Saint-Modeste, QC, Canada) composed of 70% fine peatmoss, 15% perlite, and 15% vermiculite.

Propagation phase.

Immediately after transplanting, trays were moved to a multishelf unit inside an air-conditioned growth room. The unit had four vertical compartments (126 cm long × 30 cm tall × 55 cm wide) that provided different PPFD treatments from broadband white LED fixtures (RAZRx modular array; Fluence Bioengineering Austin, TX, USA) with peak wavelengths of 450 and 660 nm, used for 24 h·d⁻¹. Two trays per cultivar and crown diameter were randomly placed inside each compartment. The PPFD treatments evaluated were 75, 150, 225, and 300 ± 5 µmol·m⁻²·s⁻¹, which resulted in DLIs of 6.5, 12.9, 19.4, and 25.9 mol·m⁻²·d⁻¹, respectively. Target PPFD setpoints were based on 12-point light maps generated using a spectroradiometer (SS-110; Apogee Instruments Inc., Logan, UT, USA) placed at midcanopy height. Air temperature was set at constant 22 °C, and RH in each compartment was maintained at approximately 90% using an ultrasonic fogger (12 Disc Mist Maker; The House of Hydro, Fort Myers, FL, USA) and solenoid valves controlled by a datalogger (CR1000X and AM16/32B multiplexer; Campbell Scientific, Logan, UT, USA) interfaced with thermistors (HygroVUE 10; Campbell Scientific). No CO₂ supplementation was provided, but CO₂ was maintained at ambient levels, as measured periodically with a portable sensor (GM70D; Vaisala Corporation, Helsinki, Finland).

Plants were subirrigated when roots were visible at the bottom of at least six cells in each tray, which occurred approximately 10 d after transplanting. A water-soluble fertilizer with micronutrients (Peter’s Professional 15N–2.2P–12.5K; ICL Specialty Fertilizers, Dublin, OH, USA) was applied at 100 mg·L⁻¹ N for the first week and increased to 150 mg·L⁻¹ N for the rest of the experiment. Flower buds and new runners were removed from all plants to encourage root growth. Plants were propagated for 28 d during each experimental replication. Average daily temperature and RH measured for each treatment during the three experimental replications are detailed in Table 1.

Table 1. Environmental parameters recorded during an experiment evaluating growth of strawberry runner tips propagated indoors under different photosynthetic photon flux density treatments or in a greenhouse evaluated as a control.

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Another group of plants were placed in the same greenhouse compartment where stock plants were grown and included as a control treatment, for which two trays of each cultivar and crown diameter were placed inside a mist compartment (1.5 m length × 1.1 m width × 1.5 height) covered with 6-mm transparent polyethylene film and 50% shadecloth. The compartment had two nozzles (CoolNet; Netafim Irrigation, Inc, Fresno, CA, USA) placed approximately 1 m above the bench surface and spaced 0.6 cm apart. Mist was provided with tap water controlled by a timer (MistTime Controlled; Dramm Corporation, Manitowoc, WI, USA). During the first 12 d, mist was provided for 10 s every 30 min during the day (0600 to 2100 HR) and every 4 h at night. Settings were subsequently adjusted so that mist was only provided for 10 s every hour during the day and every 4 h at night for the rest of the experiment. Temperature and DLI were measured with probes (107 Temperature Probe; Campbell Scientific) and quantum sensors (SQ-500-SS; Apogee Instruments, Inc.), respectively, placed outside the mist compartment. Sensors were interfaced to a datalogger (CR1000 with AM16/32B multiplexer; Campbell Scientific) and recorded data at 60-min intervals. Table 1 shows the average DLI, temperature, and RH measured in the greenhouse during the three experimental replications; DLI was calculated as 50% of the measured values to account for shade inside the mist structure.

To determine root-coverage percentage, roots of two randomly selected plants per tray were photographed at 18 and 28 d after transplanting. A digital camera (Canon PowerShot SX260 HS; Canon USA, Melville, NY, USA) mounted on a dark box similar to that described by Ghali et al. (2012) was used for this purpose. Images were subsequently analyzed following procedures by Patton et al. (2018), with modifications. Two opposite sides were photographed from each plant. The percentage of white pixels in each image was calculated using image-processing software (ImageJ Java1.48v; National Institutes of Health, Bethesda, MD, USA). An image of a white calibration disk (58.7 cm²) was used to convert white pixels to root coverage area in cm². The average of two images per plant was used as an estimation of root coverage area.

A subgroup of four plants per tray was destructively harvested at the end of the propagation phase. Before the destructive harvest, stomatal conductance and transpiration were measured between 0800 and 1300 HR on a new, fully expanded leaf from each plant using a leaf porometer (LI-600; LI-COR Biosciences, Lincoln, NE, USA). Chlorophyll concentration was subsequently measured on the same leaf using a chlorophyll meter (MC-100; Apogee Instruments). For all plants, petiole length was measured with a ruler from the substrate surface to the base of the middle leaflet. After noticing that some new shoots had died, the total number of healthy and dead shoots on each plant (>1 cm) were counted to calculate the percentage of dead shoots per plant. For the destructive harvest, shoots were separated from roots, and after the substrate was carefully washed off, root tissues were oven-dried for 72 h at 70 °C. Shoot and root DMs were subsequently determined.

Finishing phase.

The remaining four plants per tray were individually transplanted into 12.7-cm-diameter containers filled with the same substrate used for the stock plants. Plants were randomly placed on two metallic benches in the same greenhouse previously described and grown for 7 weeks. Plants were hand irrigated as needed with water-soluble fertilizer (Peter’s Professional 20N–1.3P–15.8K; ICL Specialty Fertilizers) that provided 150 mg·L⁻¹ N. Fruit from ‘Albion’ plants were first harvested approximately 4 weeks after transplanting. Fruit were then harvested weekly, and the number and fresh mass (FM) of mature fruit were recorded each time. ‘Fronteras’ produced only daughter plants due to the long photoperiod used in the greenhouse. Therefore, the total number of daughter plants produced per plant was recorded at the end of the experiment.

Experimental design and data analyses.

The propagation phase used a completely randomized block design for the indoor treatments, during which each experimental replication was regarded as a block, each with four PPFD treatments. Additionally, a greenhouse control was included for comparison. Each treatment replication had two trays per cultivar and crown diameter, each regarded as an experimental unit. Data collected from multiple plants per experimental unit were averaged and treated as a single data point. The finishing phase used a completely randomized design in which each individual plant was regarded as an experimental unit. For both phases, data were pooled among replications over time because the variances among experiments were not different and the statistical interactions among treatment and replications were not significant (P ≥ 0.05). Data were analyzed by cultivar to illustrate unique cultivar trends. For plants grown indoors, the influence of the different categorical independent variables (i.e., crown diameter and PPFD treatments) and their possible interaction on each of the continuous dependent variables were analyzed using a two-way analysis of variance. Because the crown diameter × PPFD interaction was not significant in most cases (Table 2), data were pooled for main effect treatment means. Responses to PPFD were analyzed using regression analysis (n = 12). When the regression model was significant (P ≤ 0.05), linear and quadratic fits were evaluated and selected based on the r² value for each model. To identify differences between the greenhouse control and each PPFD treatment, data were analyzed using a Dunnett’s test (P ≤ 0.05). Responses to crown diameter were analyzed using Student’s t test (n = 24). All data were analyzed using statistical software (R Studio 2023.06.1 524, ©2009 to 2020; Posit Software PBC, Boston, MA, USA).

Table 2. Significance level for variables used to evaluate growth and physiological responses of strawberry runner tips of two crown diameters (CD) (small <10 mm or large >10 mm) propagated indoors under different photosynthetic photon flux density (PPFD) treatments and finished in a greenhouse.

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Propagation phase.

The percentage of dead shoots was low in plants of both cultivars propagated under 75 μmol·m⁻²·s⁻¹ (3%) or in the greenhouse (<1%) but followed a positive quadratic response to increasing PPFD that peaked at 225 μmol·m⁻²·s⁻¹ (Fig. 1A). This suggests that plants exposed to higher PPFDs experienced radiation stress that likely led to the necrosis of new growth, plausibly attributed to a limited supply of water and nutrients during early stages of the experiment, when root growth was not sufficient to adequately sustain new shoot growth (Fig. 2). Accordingly, in a study evaluating the effect of DLI by using shadecloth to adjust PPFD, Kohler and Lopez (2021) found necrotic lesions on the surface of sage (Salvia officinalis) and spearmint (Mentha spicata) plants propagated from cuttings under DLIs ≥ 11.2 mol·m⁻²·d⁻¹, but no lesions were observed in plants propagated under DLIs ranging from 3 to 9 mol·m⁻²·d⁻¹. Miao et al. (2023) also reported a higher incidence of leaf necrosis in spinach (Spinacia oleracea) and lettuce (Lactuca sativa) seedlings propagated indoors under 300 μmol·m⁻²·s⁻¹ compared with 120 μmol·m⁻²·s⁻¹. Differences in RH may have also affected the incidence of dead shoots by increasing susceptibility to fungal growth (Table 1). This may explain why plants under 300 μmol·m⁻²·s⁻¹ had fewer dead shoots than those under 225 μmol·m⁻²·s⁻¹, which had average RH of 83% and 95%, respectively.

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Although plants propagated in the greenhouse were exposed to an average DLI of 15.1 mol·m⁻²·d^–1 (Table 1), with PPFD values reaching up to 366 μmol·m⁻²·s⁻¹, they also experienced daily periods of darkness from natural variations in day length. This large environmental difference from the plants propagated indoors may be responsible for the lower percentage of dead shoots measured in the greenhouse. Others have shown that some plants propagated under continuous lighting can develop symptoms of necrosis that are not observed under shorter photoperiods (Matsuda et al. 2014; Shibaeva et al. 2022). These symptoms are often attributed to changes in the circadian rhythm of plants that can disturb metabolic and physiological processes (Velez-Ramirez et al. 2017). It is plausible that providing a period of darkness can help mitigate the risk of developing necrosis induced by continuous lighting when strawberry plants are propagated indoors under high PPFDs.

Petioles were generally shorter in plants under higher PPFDs, which is a positive quality attribute that may help facilitate handling during and after propagation (Fig. 1B). Increases in stem and petiole elongation under lower PPFDs have been reported for various plant species (Ballaré et al. 1990; Hernández and Kubota 2014; Lopez and Runkle 2008; Park et al. 2011; Pramuk and Runkle 2005; Torres and Lopez 2011) and are often attributed to a shade-avoidance response that enables plants to increase the amount of light they can intercept (Kozuka et al. 2010). Interestingly, plants in the greenhouse grew longer petioles than those propagated indoors, which may have been caused by differences in the day/night temperature (DIF) between the two environments. Although temperature in the greenhouse was set to a constant 24 °C, the plants were exposed to an average positive DIF of 3 °C, driven by our limitations to fully control the greenhouse environment. In contrast, plants indoors were grown under a DIF of approximately 0 °C. As indicated by Moe and Heins (2000), positive DIF can induce petiole elongation in several plant species. Others have attributed this response to changes in the concentration of hormones such as gibberellin, which affect cell-wall-related genes when days are warmer than nights (Ohtaka et al. 2020). Another plausible explanation for the difference in petiole length between plants propagated in the two environments could be the difference in far-red radiation, as only the plants in the greenhouse were exposed to far-red radiation from sunlight, which may have stimulated petiole elongation (Tan et al. 2022).

Shoot and root DM of both cultivars followed a positive linear response to increasing PPFD, suggesting that further increases in PPFD could have continued to increase biomass production (Fig. 1C, 1D). ‘Fronteras’ produced 44% more shoot DM under 300 μmol·m⁻²·s⁻¹ compared with plants in the greenhouse and over 30% more root DM under 225 or 300 μmol·m⁻²·s⁻¹. Similarly, ‘Albion’ under 150 or 300 μmol·m⁻²·s⁻¹ produced 29% or 37% more root DM than plants in the greenhouse, respectively. Our results are consistent with the findings of others who have reported increases in shoot and root DM of various plant species under higher PPFDs. As explained by Currey and Lopez (2015), higher PPFDs during vegetative propagation increase photosynthetic capacity and carbohydrate availability, typically resulting in more growth.

Root coverage of ‘Fronteras’ followed a linear increasing response to increasing PPFD, but no treatment differences were measured for ‘Albion’ at 18 d (Fig. 3A). However, at 28 d, root coverage of both cultivars followed a quadratic response that peaked at 300 µmol·m⁻²·s⁻¹ for ‘Fronteras’ and at 225 µmol·m⁻²·s^–1 for ‘Albion’ (Fig. 3B). At 18 d, only ‘Fronteras’ under 300 µmol·m⁻²·s⁻¹ had greater root coverage than plants in the greenhouse. However, root coverage at 28 d was generally higher in plants of both cultivars propagated indoors under ≥150 μmol·m⁻²·s⁻¹ compared with the greenhouse. Our results indicate that root coverage plateaued sometime during the experiment, because side-by-side (non-statistical) comparisons at 18 and 28 d showed relatively similar treatment means. Interestingly, however, root coverage of plants under 300 µmol·m⁻²·s⁻¹ was 50% to 35% greater at 18 d than that measured in plants under 75 µmol·m⁻²·s⁻¹ at 28 d. Taken together, these findings suggest that plants under 300 µmol·m⁻²·s⁻¹ could have been transplanted at 18 d, which would drastically reduce the propagation cycle and, thus, enable faster turnover in indoor propagation systems. Similar findings were reported by Gómez et al. (2021), who showed that the biomass of blueberry microcuttings was higher under 140 µmol·m⁻²·s⁻¹ at 4 weeks compared with that measured at 8 weeks under 35 µmol·m⁻²·s⁻¹. The authors concluded that higher PPFDs could significantly increase turnover during indoor propagation. Another potential advantage of shortening the rooting cycle by propagating strawberry runner tips under 300 µmol·m⁻²·s⁻¹ would be to minimize exposure to the aforementioned high-radiation stress, especially considering that stress responses were more pronounced toward the end of the experiment (data not shown).

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Chlorophyll concentration of both cultivars linearly decreased with increasing PPFD (Fig. 4A), and ‘Albion’ plants under 75 or 150 μmol·m⁻²·s⁻¹ had higher chlorophyll concentration than those propagated in the greenhouse. Similar to our results, Zheng et al. (2019) found that chlorophyll concentration of strawberry runner tips propagated under 210 μmol·m⁻²·s^–1 was 32% lower than that of plants under 30 μmol·m⁻²·s⁻¹. Other studies evaluating the effect of PPFD during cutting propagation have shown decreases in chlorophyll concentration with increasing PPFD (Sato et al. 2015; Staton and Gómez 2024). In our study, some leaves of plants under 225 or 300 μmol·m⁻²·s⁻¹ were red (Fig. 5), which is consistent with observations made by others who have attributed the response to an increase in anthocyanin concentration as a photoprotective response to mitigate high-radiation stress (Gómez et al. 2021; Kelly et al. 2020; Staton and Gómez 2024; Zheng et al. 2019). Increases in anthocyanin concentration are usually accompanied by a decrease in chlorophyll concentration in leaves (Shao et al. 2014), which may explain the results measured in our study.

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Both stomatal conductance and transpiration followed positive quadratic responses to increasing PPFD (Fig. 4B, 4C). Zheng et al. (2019) reported similar results and attributed their findings to mild photoinhibition caused by increasing PPFD when propagating strawberry runner tips indoors. Accordingly, other studies have shown that although higher PPFDs generally support efficient gas exchange, extreme increases in PPFD can reduce gas exchange during vegetative propagation caused by high-radiation stress (Lee et al. 2007). In our study, results for stomatal conductance and transpiration were likely associated with the aforementioned stress, which may have caused partial stomatal closure under higher PPFDs and, thus, reduced the ability of plants to efficiently transpire and exchange CO₂ and water vapor with the atmosphere.

Finishing phase.

There were no carryover growth or yield responses to PPFD during the finishing phase (Fig. 6), which was surprising, considering that both shoot and root DM during propagation linearly increased with higher PPFD (Fig. 1C, 1D). Hochmuth et al. (2006) showed that increases in biomass during the propagation of strawberry plants tend to result in earlier fruit production after transplant, which ultimately increase yield. However, our general findings suggest that factors beyond PPFD need to be adjusted during propagation to increase subsequent growth and yield of strawberry plants. Yoshida et al. (2016) and Tsuruyama and Shibuya (2023) explained that fruit production of strawberry can increase by inducing early flower bud formation during propagation. Accordingly, Park et al. (2023) found that extending the photoperiod during propagation from 12 to 16 h enabled ‘Albion’ plants to flower faster and produce more fruit, but increasing PPFD had minimal effects on yield, which is consistent with our results. For ‘Elan’ and ‘Yotsuboshi’, which are two long-day strawberry cultivars, Tsuruyama and Shibuya (2018, 2023) showed that providing far-red radiation coupled with extending the photoperiod during indoor propagation promoted flower development and increased subsequent fruit yield in the greenhouse. Temperature is another factor that is known to affect flower development in strawberry plants, and some long-day cultivars require cooler temperatures than short-day plants to induce flowering (Sønsteby and Heide 2008). Therefore, in addition to providing adequate PPFD, propagating strawberry plants under photoperiods, light qualities, and temperatures that stimulate flower development may help increase yield during subsequent fruiting stages. However, recommendations will depend on the cultivar being grown and its specific environmental requirements (Durner et al. 2002; Xu and Hernández 2020).

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The general lack of treatment response during the finishing phase in our study differs from the findings of others (Fig. 6). For example, Walters and Lopez (2022) reported increases in yield and flavor quality during a greenhouse finishing phase of basil (Ocimum basilicum) seedlings propagated indoors under increasing PPFD. Similar results were reported by Givens et al. (2023) and Kong et al. (2018) when evaluating the effect of PPFD on lettuce and pea (Pisum sativum) plants propagated indoors, respectively, demonstrating that higher PPFDs in propagation can increase subsequent growth and quality in seedlings. In general, these carryover effects have been attributed to changes in the photosynthetic capacity of plants when exposed to higher PPFDs. A plausible explanation for the lack of treatment differences during the finishing phase in our study could be attributed to the abrupt transition from indoors to the highly variable greenhouse environment, which may have minimized initial growth differences established during propagation (Fig. 1). For example, various studies have shown that fluctuations in environmental factors such as temperature, RH, and light intensity, quality, and duration can minimize growth differences that had been previously measured in response to preimposed environmental treatments (Hidaka et al. 2013; Park et al. 2023). In addition, it is likely that the energy allocation of plants toward fruit production increased once they were transferred to the greenhouse, which may have affected growth of vegetative tissues and, thus, could explain the general lack of differences in response to PPFD. Accordingly, Hidaka et al. (2015) showed that during the fruiting phase, most photosynthates are translocated to strawberry fruit, which minimized differences in subsequent vegetative growth. Their findings were attributed to the higher sink strength of fruits compared with shoots and roots (Hidaka et al. 2014).

Interestingly, ‘Albion’ plants in the greenhouse produced more fruit FM than those propagated indoors under 225 or 300 μmol·m⁻²·s⁻¹, which was likely attributed to the stress that plants in those two treatments experienced (Fig. 6D). Accordingly, Lima-Melo et al. (2019) showed that stress and photoinhibition caused by high PPFDs can negatively affect fruit yield of strawberry plants.

Effects of crown diameter.

Shoot and root DM of both cultivars were higher in plants with large crowns, but the opposite trend was measured for stomatal conductance and transpiration. When using large crowns, ‘Albion’ produced longer petioles, and ‘Fronteras’ had higher chlorophyll concentration, but no other effects of crown diameter were measured in the propagation phase (Table 3). Similar to our findings, Cocco et al. (2011) found that runner tips with crown diameters between 5.6 and 7.0 mm produced more vigorous transplants with higher shoot and root DM than those with crown diameters ranging from 2.0 to 5.5 mm. These results are likely attributed to the fact that runner tips are able to store more carbohydrate reserves in large crowns (Fridiaa et al. 2016), which helps support initial growth at the beginning of the propagation phase and likely enables plants to get established faster (Zhu et al. 2024).

Table 3. Means for variables used to evaluate growth and physiological responses of strawberry runner tips of two crown diameters (small <10 mm or large >10 mm) propagated indoors and finished in a greenhouse.

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The higher stomatal conductance and transpiration measured in plants with small crowns may be attributed to their smaller size (Table 3). Our results show that, in general, runner tips with small crowns tend to produce less shoot DM and shorter petioles during propagation, which suggests that the leaves are also smaller. Other studies have shown that to facilitate efficient gas exchange, smaller leaves tend to have more stomata per unit area than do larger leaves of the same species, which can increase stomatal conductance and transpiration (Sack and Buckley 2016). The higher chlorophyll concentration in ‘Fronteras’ with large crowns may be related to the higher shoot DM and larger leaves produced per plant, which may have enabled higher whole-plant photosynthesis and, thus, contributed to the slightly higher concentration of chlorophyll. Accordingly, Ullah et al. (2024) found a positive correlation between chlorophyll concentration and shoot DM of strawberry plants.

During the finishing phase, the effect of crown diameter was only significant for fruit number and total fruit FM of ‘Albion’ (Table 2), both of which were higher in plants with larger crowns (Table 3). These findings are consistent with those of others who have reported higher fruit yield when strawberry plants are started from runner tips with large crowns (Bish et al. 2002; Takeda and Newell 2007). Fagherazzi et al. (2021) found that plants propagated from crowns >10 mm produced 18% more fruit and 27% higher fruit FM than those started from crowns <10 mm. Similarly, Torres Quezada et al. (2015) reported 8% higher fruit FM from the first harvest in strawberry plants propagated from crowns >10 mm compared with those <10 mm. In addition, at the end of the production cycle, the authors found no differences in shoot DM between plants started from different crown diameters, which is consistent with our results (Torres Quezada et al. 2015). Increases in yield when strawberry plants are started from larger crown diameters have been attributed to the presence of more buds that can differentiate into flowers and fruit.

The lack of response to crown diameter in the number of daughter plants produced by ‘Fronteras’ could be related to the duration of the finishing phase in our study (7 weeks), which was likely too short to affect daughter-plant production. Accordingly, Shi et al. (2021) found that the maximum productivity of strawberry daughter plants occurred approximately 8 weeks after starting an experiment evaluating different harvest times. Similar to our findings, Lisiecka (2009) reported that the number of daughter plants produced per plant was unaffected by crown diameter.