Materials and methods

Two experimental runs were conducted in this study, which consisted of two phases, an acclimation phase indoors where treatments were imposed as described subsequently, and a finishing phase in a greenhouse to evaluate carryover treatment effects. For the acclimation phase, shoot-tip cuttings of chrysanthemum (Chrysanthemum indicum) ‘Crystal Bronze Bicolor’ (5-cm stems, four to five fully expanded leaves) and elatior begonia (Begonia ×hiemalis) ‘Dark Britt’ (∼2.5-cm stems and one fully expanded leaf) were received from a commercial supplier (Dümmen Orange, Columbus, OH, USA) on 24 Oct 2023 and 14 Nov 2023 during the first and second run, respectively. Cuttings were immediately stuck into industry-standard, 72-cell propagation trays (41 mL, individual cell volume; T.O. Plastics, Inc., Clearwater, MN, USA) cut into 3 × 3 partial trays filled with horticultural grade substrate (Berger BM2 Seed Germination; Berger, Saint-Modeste, Canada) composed of 70% fine peatmoss, 15% perlite, and 15% vermiculite. The substrate had an electrical conductivity (EC) of 0.8 dS·m⁻¹, as measured using a portable pH/EC meter (HI9813-6; Hanna Instruments, Carrollton, TX, USA) following the pour-through method. Immediately after sticking, half of the trays were manually irrigated with a complete fertilizer solution (15N–2.2P–12.5K with micronutrients; Peter’s Professional; ICL Specialty Fertilizers, Summerville, SC, USA) dissolved in tap water (0.8 dS·m⁻¹ EC, 7.3 pH, and 31.2 mg·L⁻¹ calcium carbonate) that provided 100 mg·L⁻¹ N, resulting in a substrate EC of 1.1 dS·m⁻¹. The remaining trays were irrigated with reverse osmosis (RO) water, resulting in a substrate EC of 0.7 dS·m⁻¹. After allowing trays to drain for 20 min, cuttings were moved to a walk-in growth chamber (C6 Control System with ECoSys^TM Software; Environmental Growth Chambers, Chagrin Falls, OH, USA) with two opposite shelving units, each with an upper and a lower compartment (180 cm long × 80 cm wide) lined with insulation foam at the bottom. Two broadband white LED fixtures (RAY66 PhysioSpec Indoor^TM; Fluence Bioengineering, Austin, TX, USA) with peak wavelengths of 446, 599, and 664 nm were placed in each of the four compartments and used for 24 h·d⁻¹, which provided 19% blue, 41% green, and 39% red, and 1% far-red light.

Four trays with cuttings of each species and fertilizer treatment were placed in each compartment, which provided a PPFD at canopy height of 70 ± 2.5 µmol·m⁻²·s⁻¹. Two trays per species and fertilizer treatment were then raised in each compartment by placing them onto 9.4-in tall cavity support trays, which increased the PPFD at canopy level to 140 ± 2.8 µmol·m⁻²·s⁻¹. Target PPFDs were based on 12-point light maps generated using a spectroradiometer (SS-110; Apogee Instruments Inc., Logan, UT, USA). Air temperature, relative humidity (RH), and carbon dioxide (CO₂) concentration were set at constant 22 °C, 70%, and 550 µmol·mol⁻¹, respectively. Air temperature and RH within each compartment were monitored with dataloggers (HOBO UX100-023; Onset Computer Corporation, Bourne, MA, USA) placed at canopy height. Values were measured every 10 s and recorded at 60-min intervals. Average CO₂ concentration was measured with a CO₂ sensor (GMW86P; VAISALA, Vantaa, Finland) and logged every 15 min using a built-in datalogger (DL1 Datalogger; Environmental Growth Chambers).

Trays were covered with clear plastic, vented humidity domes (54 cm × 28 cm × 15 cm; Acro Dome, Acro Plastics, LTD, Edmonton, Canada) to maximize RH during root initiation. Cuttings were manually misted with RO water twice daily until initial root emergence, which occurred 7 and 14 d after sticking for chrysanthemum and begonia, respectively. Once root initials were visible, domes were removed and cuttings were manually irrigated once using either fertilizer solution or tap water, as previously described. Chrysanthemum and begonia were acclimated indoors for 14 and 21 d, respectively, followed by a destructive harvest of five cuttings per tray. The remaining four cuttings per tray were immediately transferred to a common glass-glazed greenhouse located in West Lafayette, IN, USA (lat. 40°N) and grown for 7 d, followed by a destructive harvest to assess carryover treatment effects, which occurred 21 and 28 d after sticking for chrysanthemum and begonia, respectively.

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, Canada). Supplemental lighting was delivered by 1000-W high-pressure sodium lamps (P.L. Light Systems Inc., Beamsville, Canada) used for 16 h·d⁻¹ (0500 to 1900 HR) providing a PPFD of ∼150 µmol·m⁻²·s⁻¹. Temperature and RH setpoints in the greenhouse were kept at 24/22 °C (day/night) and 65%, respectively. In the greenhouse, cuttings were manually irrigated with acidified tap water (twice) or with the same fertilizer solution previously described (once). The average air temperature, RH, and CO₂ concentration for the acclimation phase indoors during the two experimental runs were (mean ± SD) 22.3 ± 2.1 °C, 72% ± 4%, 562 ± 70 µmol·mol⁻¹, respectively. Average air temperature, RH, and daily light integral (DLI) for the finishing phase in the greenhouse during the two experimental runs were 24.3 ± 1.1 °C, 50 ± 9%, and 15 mol·m⁻²·d⁻¹, respectively.

Data collection and analyses

For each harvest, shoot height of chrysanthemum was measured from the substrate surface to the apical meristem. Chlorophyll concentration was measured for both species before the end of the acclimation phase using a chlorophyll meter (MC-100; Apogee Instruments). However, chlorophyll concentration was only measured for begonia before the end of the finishing phase. The length of the longest root and number of visible roots (>2 mm) were recorded during each harvest after the substrate was carefully washed off the roots. Shoot and root dry weight (DW) were measured by placing bagged tissues in a forced-air oven for 5 d at 70 °C, and samples were subsequently weighed using an electronic balance.

Data were analyzed per species as a two (fertilizer application) × two (PPFD treatment) factorial with four replications in space (compartment) and two replications in time. For each replication in time, tray placement was randomized within each compartment, which held two trays with cuttings of each species for each factor described earlier. For each harvest, data from all cuttings per tray were averaged and treated as a single data point per replication. Data were pooled between replications over time because the variances between experimental runs were not different and the statistical interactions between treatment and replications were not significant (P ≥ 0.05). Data were subjected to analyses of variance using a statistical software (JMP Pro, version 16; SAS Institute Inc., Cary, NC, USA). Because the fertilizer application × PPFD interaction and main effect of fertilizer application were not significant in most cases (Table 1), data are only presented for the main effect of PPFD and compared using Tukey’s honestly significant difference test (P ≤ 0.05) (n = 16).

Table 1.

Significance level for variables used to evaluate quality and growth of unrooted cuttings acclimated under different fertilizer and photosynthetic photon flux density (PPFD) treatments indoors and finished in a greenhouse.

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Results and discussion

Fertilizer application had no growth effects during the indoor acclimation phase, except for shoot DW of begonia (Table 1), for which unfertilized cuttings had 8% higher shoot DW than those treated with fertilizer (data not shown). Our findings differ from those of Budiarto et al. (2006), who reported increases in root growth of chrysanthemum cuttings with higher N concentrations. Similarly, Santos et al. (2008) showed that cuttings of petunia (Petunia ×hybrida) that received fertilizer had higher root DW and produced more and longer roots than those treated with tap water. In another study, Santos et al. (2011) showed that constant application of a complete fertilizer solution through mist provided rooting advantages when propagating petunia cuttings in a greenhouse. However, Budiarto et al. (2006) found no differences in root growth when comparing different frequencies of fertilizer application. The general lack of response to fertilizer application in our study is likely attributed to the fact that fertilizer was only applied twice during the acclimation phase. The average substrate EC measured after the second fertilizer application from a subgroup of fertilized and unfertilized trays were 1.4 and 0.8 dS·m⁻¹, respectively, which may not have been sufficiently different to affect rooting and growth. In addition, cuttings in our study experienced limited leaching of nutrients due to the limited number of irrigation events and from the lack of mist applied to the substrate, which is common during greenhouse propagation and may explain differences between our findings and those of Budiarto et al. (2006) and Santos et al. (2011). Considering that cuttings of both species were visually healthy and had no signs of nutrient deficiency, our findings suggest that when using a substrate with a fertilizer starter charge providing an EC of 0.8 dS·m⁻¹, they can be successfully acclimated in VIP systems without additional fertilizer. However, the fertilization management of stock plants will ultimately determine the need to apply fertilizer during acclimation, given that cuttings with low initial tissue–nutrient concentrations may benefit from complete fertilizer applications, regardless of propagation environment (Santos et al. 2011).

Chlorophyll concentration was the only variable that showed a fertilizer × PPFD interaction (P < 0.0001) (Table 1), for which unfertilized chrysanthemum cuttings had the highest and lowest chlorophyll concentration after the acclimation phase under the higher and lower PPFD, respectively (39.0 and 24.2 μmol·m⁻², respectively) (data not shown). For begonia, cuttings under the higher PPFD had 20% less chlorophyll concentration but produced 12% more shoot DW than those under the lower PPFD (Table 2). However, no differences in root DW were measured for begonia under the two PPFD treatments, whereas that of chrysanthemum was almost double under the higher than the lower PPFD. Similarly, chrysanthemum cuttings under the higher PPFD produced 39% more shoot DW than those under the lower PPFD, but cuttings were 5% shorter. There was no response to PPFD for the length of the longest root or the number of visible roots per cutting for either species.

Table 2.

Quality and growth responses of unrooted cuttings acclimated under different photosynthetic photon flux densities (PPFD) indoors and finished in a greenhouse.

View Table

Our findings are similar to those reported by Olschowski et al. (2016), who showed that cuttings of Calibrachoa under a PPFD of 80 µmol·m⁻²·s⁻¹ were shorter and had more shoot and root biomass than those propagated under 40 µmol·m⁻²·s⁻¹. Similarly, Park et al. (2011) and Gil et al. (2020) concluded that higher PPFD during acclimation improved rooting of rose (Rosa hybrida) and chrysanthemum cuttings, respectively, compared with lower PPFD. As explained by Currey and Lopez (2015), higher PPFD increases photosynthetic capacity and carbohydrate availability of cuttings. However, their findings are based on responses after the initial acclimation phase of cuttings, when susceptibility to stress caused by high PPFD is typically lower (Currey and Lopez 2015). Although our results show that a PPFD of 140 µmol·m⁻²·s⁻¹ is suitable to start cuttings of chrysanthemum and begonia in VIP systems (Table 2), further studies are needed to identify the ideal PPFD to maximize root development and growth without causing stress. This is important, considering that PPFDs that result in DLIs ≥10 mol·m⁻²·d⁻¹ can be detrimental during acclimation due to excessive evapotranspiration and subsequent wilting of cuttings (Loach 1988). In addition, higher PPFDs during acclimation can cause a red coloration in the foliage of some species, which has been reported by others (Gómez et al. 2021) and is thought to be the result of a stress-induced increase in anthocyanin. Accordingly, leaves of begonia cuttings under the higher PPFD were visibly red compared with those under the lower PPFD (data not shown), which may explain their lower chlorophyll concentration.

The only response to fertilizer application during the finishing phase was measured for root DW of chrysanthemum (Table 1), for which unfertilized cuttings produced 24% more root DW than those treated with fertilizer (data not shown). During the finishing phase, there were no differences in shoot height of chrysanthemum or chlorophyll concentration of begonia in response to PPFD (Table 2). This corresponds with a visual attenuation of the aforementioned reddening observed in leaves of begonia cuttings acclimated under the higher PPFD. Gómez et al. (2021) also showed that changes in leaf color and differences in chlorophyll concentration in response to PPFD during indoor acclimation were no longer significant after a carryover phase in a greenhouse.

Chrysanthemum that had been acclimated under the higher PPFD produced 9% and 14% more shoot and root DW, respectively, than those under the lower PPFD (Table 2). However, no treatment differences were measured for begonia. The different responses to PPFD between the two species during the finishing phase may be attributed to their different light requirements and rooting times during acclimation. Chrysanthemum cuttings require more light and generally root faster than those of elatior begonia (Nau et al. 2021). Thus, they were likely able to use the higher PPFD immediately after root emergence, which helped increase biomass production from that point forward. In contrast, the begonia cultivar used in our study is sometimes considered slow to root, which, coupled with its lower light requirement, could explain its limited response to PPFD because active growth of cuttings typically increases after initial root development.

In conclusion, when using a substrate with a fertilizer starter charge, additional fertilizer applications during cutting acclimation in VIP systems may not benefit shoot and root growth of ornamental herbaceous species under conditions similar to those used in our study. However, the nutritional status of cuttings and that of stock plants must be considered before making decisions about fertilizer use. Furthermore, high-light-requiring species such as chrysanthemum are more likely to benefit from higher PPFD during indoor acclimation than those that require less light, such as begonia.