Materials and Methods

Propagation environment and culture

All cuttings were placed in a glass-glazed greenhouse under a 16-h photoperiod, with air and substrate temperature set points of 23 ± 1 °C, and a DLI of ≈5 mol·m⁻²·d⁻¹ was maintained for callusing using a combination of shade cloth and supplemental lighting from 1000-W high-pressure sodium (HPS) lamps (e-system HID; PARSource, Petaluma, CA) that delivered a supplemental photosynthetic photon flux (PPF) of ≈30 µmol·m⁻²·s⁻¹ at plant height [as measured with a quantum sensor (LI-COR Biosciences, Lincoln, NE)] when outdoor irradiance was less than 250 μmol·m⁻²·s⁻¹ from 0600 to 2200 hr. The substrate temperature was maintained by using bench-top rubber tubing with circulating hot water (49 °C) controlled by a substrate thermistor probe (BioTherm Benchwarmer Kit; TrueLeaf Technologies, Petaluma, CA). Beginning at placement of cuttings in the propagation system, mist was applied for 4 s every 20 min beginning and ending with the photoperiod consisting of reverse osmosis water supplemented with a complete water-soluble fertilizer (Jack’s LX 16N–0.94P–12.3K Plug Formula for High Alkalinity Water; JR Peters Inc., Allentown, PA) and micronutrient supplement (Compound 111; Scotts, Marysville, OH) providing with each misting event (mg·L⁻¹): 60 N, 6 P, 47 K, 11 Ca, 6.2 Mg, 0.3 B, 0.1 Cu, 1.5 Fe, 0.4 Mn, 0.09 Mo, and 0.2 Zn. Thermistors (External Temperature Sensor; Spectrum Technologies, Plainfield, IL) recorded air and substrate temperatures every 30 s and averages were logged every 15 min by a data logger (Watchdog 2800 Weather Station; Spectrum Technologies), and the daily averages were automatically calculated and logged. Amplified quantum sensors (SQ-212; Apogee Instruments, Logan, UT) measured PPF every 30 s under each lighting treatment, and the average of each sensor was logged every 15 min by a data logger (Watchdog 2800 Weather Station) and the DLI was automatically calculated and logged. Environmental data during callusing are reported in Table 1.

Table 1.Average daily greenhouse air and substrate temperatures and daily light integral (DLI) during callusing of new guinea impatiens ‘Celebration Pink’, geranium ‘Fantasia Dark Red’, and petunia ‘Suncatcher Midnight Blue’ preceding placement under different DLI treatments during root development.

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Supplemental lighting treatments

After 7 d of callusing, 105 cuttings of each species were placed under one of three treatments that were created using either no shade or fixed-woven shade cloth providing ≈38% or 86% shade (XLS F-14 or F-16; Ludvig Svensson, Charlotte, NC), respectively, under ambient daylight with supplemental PPF of ≈93.8 ± 10.9, 49.2 ± 4.5, and 27.8 ± 1.7 μmol·m⁻²·s⁻¹, respectively, at plant height [as measured with a quantum sensor (LI-COR Biosciences)], respectively, from 0600 to 2200 hr (Table 2). Supplemental light was delivered from HPS lamps (e-system HID, PARsource). An automatic woven shade curtain (OLS 50; Ludvig Svensson) was employed when the outdoor light intensity exceeded ≈1000 μmol·m⁻²·s⁻¹ throughout the study to prevent leaf scorch. Air and substrate temperature and DLI for each DLI treatment were measured as previously described and are reported in Table 2.

Table 2.Average daily greenhouse air and substrate temperatures and daily light integral (DLI) during root development of new guinea impatiens ‘Celebration Pink’, geranium ‘Fantasia Dark Red’, and petunia ‘Suncatcher Midnight Blue’ grown under 0%, 38%, or 86% shade under ambient daylight supplemented with ≈93.8 ± 10.9, 49.2 ± 4.5, and 27.8 ± 1.7 µmol·m⁻²·s⁻¹, respectively, delivered from high-pressure sodium lamps from 0600 to 2200 hr.

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On transfer of all cutting species to DLI treatments, misting frequency was reduced to 4 s every 30 min to begin and end with the photoperiod. Four days after the placement of cuttings under DLI treatments, the use of mist was discontinued, and cuttings were hand irrigated daily with acidified water supplemented with a combination of two water-soluble fertilizers (3:1 mixture of 15N–2.2P–12.5K and 21N–2.2P–16.6K, respectively; Everris, Dublin, OH) to provide the following (mg·L⁻¹): 200 N, 26 P, 163 K, 50 Ca, 20 Mg, 1.0 Fe, 0.5 Mn. 0.5 Zn, 0.2 Cu, 0.2 B, and 0.1 Mo. Irrigation water was supplemented with 93% sulfuric acid (Brenntag, Reading, PA) at 0.08 mL·L⁻¹ to reduce alkalinity to 100 mg·L⁻¹ and pH to a range of 5.8 to 6.2.

Results

New guinea impatiens.

Stem length and node number increased by 51% (1.3 cm) and 45% (1.1 nodes) from −1 to 14 DAT, but were unaffected by the different DLI treatments (data not shown). While DLI did not have an impact on stem caliper up to 8 DAT across DLIs, stem caliper increased by 26% (1.3 mm) as DLI increased from 2.0 to 13.4 mol·m⁻²·d⁻¹ 14 DAT (data not shown). Beginning at 8 DAT, the RDM, SDM, and LDM increased linearly with DLI within DAT up to 211% (44.9 mg), 104% (23.0 mg), and 99% (102.9 mg), respectively, by 14 DAT as DLI increased from 2.0 to 13.4 mol·m⁻²·d⁻¹ (Fig. 1A, D, and G). From −1 to 5 DAT, the RMR increased for all cutting but was unaffected by DLI; from 8 to 14 DAT, RMR increased by 15% (0.03) to 42% (0.02), respectively, as DLI increased from 2.0 to 13.4 mol·m⁻²·d⁻¹. Alternatively, the LMR of new guinea impatiens cuttings was unaffected by DLI and decreased from −1 to 8 DAT, and as the DLI increased from 2.0 to 13.4 mol·m⁻²·d⁻¹, LMR increased by 8% to 10% (0.05‒0.07) from 11 or 14 DAT. The SMR was unaffected by DLI and decreased by 39% (0.09) from −1 to 14 DAT (data not shown). Total leaf area, LAR, and SLA were affected differently over time by DLI (Fig. 2A, D, and E). For example, leaf area increased by 109% (19.2 cm²) from −1 to 11 DAT and was unaffected by DLI; however, 14 DAT, leaf area increased by 30% (11.5 cm²) as DLI increased from 2.0 to 13.4 mol·m⁻²·d⁻¹. Over time, the LAR increased for cuttings under the low-DLI treatment and decreased for cuttings under the high-DLI treatment, resulting in cuttings with 12% to 39% (0.03‒0.10 cm²·mg⁻¹) lower LAR 5 to 14 DAT. Similarly, while the SLA was stable or slightly declined for cuttings under the high-DLI treatment, it increased for cuttings under the low-DLI treatment. From 2 to 14 DAT, the SLA was 8% to 34% (0.02‒0.12 cm²·mg⁻¹) lower for cuttings under 13.4 mol·m⁻²·d⁻¹ compared with those under 2.0 mol·m⁻²·d⁻¹.

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Beginning 8 DAT, R_d increased by 64% to 83% (0.3‒0.4 μmol·m⁻²·s⁻¹) within DAT as DLI increased from 2.0 to 13.4 mol·m⁻²·d⁻¹ (Fig. 3A). The α of new guinea impatiens was unaffected by DLI (data not shown). The P_gmax of cuttings increased by 60% to 165% (2.7‒6.7 μmol·m⁻²·s⁻¹ CO₂) as DLI increased from 2.0 to 13.4 mol·m⁻²·d⁻¹. Similarly, the LCP and LSP increased by 80% to 117% (5.1‒5.8 μmol·m⁻²·s⁻¹ PPF) and 51% to 237% (127‒318 μmol·m⁻²·s⁻¹ PPF) as DLI increased from 2.0 to 13.4 mol·m⁻²·d⁻¹, respectively, from 8 DAT onward. Furthermore, starch concentration increased with DLI between 313% and 946% (28.2 and 49.8 mg·g⁻¹) from 5 to 14 DAT.

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Geranium.

Stem length, stem caliper, and node number were unaffected by DLI during propagation and increased by 27% (1.5 cm), 8% (0.5 mm), and 35% (1.8 nodes), respectively, as DAT increased from −1 to 14 (data not shown). As the DLI increased from 2.0 to 13.0 mol·m⁻²·d⁻¹, the RDM of geranium cuttings increased by 60% (1.0 mg) 2 DAT to 76% (26.4 mg) 14 DAT, while SDM and LDM were generally unaffected by DLI and increased by 45% (39.0 mg) and 65% (197.1 mg), respectively, as DAT increased (Fig. 1B, E, and H). Across DLIs, the RMR increased until 8 DAT and then stabilized. In addition, RMR increased with DLI from 2 DAT onward. While the LMR decreased similarly for cuttings under each DLI until 5 DAT, LMR then increased linearly as DLI increased from 2.4 to 13.0 mol·m⁻²·d⁻¹ from 8 to 14 DAT. While the LA of geranium cuttings increased by 107% (51.4 cm²) as DAT increased regardless of DLI, the increase in LAR and SLA was greater for cuttings grown under 2.0 mol·m⁻²·d⁻¹ compared with cuttings grown under 13.0 mol·m⁻²·d⁻¹ starting 8 and 11 DAT, respectively (Fig. 2B, E, and H).

The R_d of geranium cuttings was unaffected by DLI from −1 to 8 DAT, but increased by up to 58% (0.6 μmol·m⁻²·s⁻¹) with increasing DLI on 14 DAT (Fig. 3B). Alternatively, P_gmax increased by 55% to 76% (3.4–4.7 μmol·m⁻²·s⁻¹ CO₂) from 8 to 14 DAT. Similarly, the LCP and LSP increased by up to 62% (11.8 μmol·m⁻²·s⁻¹ PPF) beginning 8 DAT and 90% (254.8 μmol·m⁻²·s⁻¹ PPF) 11 DAT, respectively. The α was unaffected by the interaction of DLI and DAT (0.08). Beginning at 2 DAT, starch concentration increased with DLI by up to 520% (19.8 mg·g⁻¹).

Petunia.

The stem length, stem caliper, and node number of petunia were unaffected by DLI during propagation and increased by 80% (2.4 cm), 8% (0.08 mm), and 133% (4.0 nodes), respectively, from −1 to 14 DAT (data not shown). The RDM, SDM, and LDM increased across all DLIs as DAT increased throughout the experiment, though to different magnitudes. For example, at 14 DAT, the RDM, SDM, and LDM increased by 195% (19.5 mg), 118% (24.3 mg), and 122% (70.1 mg), respectively, as DLI increased from 2.3 to 14.2 mol·m⁻²·d⁻¹ (Fig. 1C, F, and I). Though, the SMR was unaffected by DLI and decreased by 42% (0.09) during propagation as DAT increased, the RMR increased by up to 85% (0.10) and the LMR decreased by up to 15% (0.08) as DLI increased from 2.3 to 14.2 mol·m⁻²·d⁻¹ 8 DAT. Leaf area increased for all cuttings regardless of DLI from −1 to 8 DAT, but 11 and 14 DAT saw increases of 36% and 112% (7.3 and 29.2 cm²), respectively, with an 11.9 increase in DLI (Fig. 2C). Beginning at 2 DAT, LAR decreased by 8% to 29% (0.02‒0.07 cm²·mg⁻¹) while the SLA decreased by 5% to 22% (0.02‒0.09 cm²·mg⁻¹) as the DLI increased from 2.3 to 14.2 mol·m⁻²·d⁻¹.

Although R_d increased by 48% (0.5 μmol·m⁻²·s⁻¹) with DLI at 14 DAT, it was generally unaffected by DLI or DAT (Fig. 3C). As DAT increased, P_gmax of cuttings propagated under 2.3 mol·m²·d⁻¹ decreased by up to 47% (12.2 μmol·m⁻²·s⁻¹ CO₂) compared with cuttings propagated under 14.2 mol·m⁻²·d⁻¹. The α of petunia cuttings was unaffected by DLI during propagation (data not shown). While there was no clear impact of DLI or DAT on the LCP of petunia cuttings, the LSP increased as DLI increased from 2.4 to 14.2 mol·m⁻²·d⁻¹ by 46% (309.6 μmol·m⁻²·s⁻¹ PPF), 124% (584.6 μmol·m⁻²·s⁻¹ PPF), and 73% (373.5 μmol·m⁻²·s⁻¹ PPF) on 2, 8, and 14 DAT, respectively. Alternatively, starch concentrations consistently increased linearly with DLI from 5 DAT onward.

Discussion

For all three species, biomass accumulation in roots, stems, and leaves was enhanced with increasing DLI (Fig. 1A−I). Biomass allocation patterns for impatiens, geranium, and petunia appeared to have plasticity, and plants acclimated in response to DLI during root development. Previously, Currey and Lopez (2012) reported that biomass allocation of new guinea impatiens ‘Magnum Salmon’ into roots increased with increasing DLI, whereas the LMR decreased; however, SMR was unaffected by DLI during root development. When our data from this research are taken together with previous research, biomass accumulation for leaves, stems, and roots generally increases with increasing DLI for cuttings during propagation. However, the proportion of biomass allocation into roots increases at the expense of allocation into leaves, as the SMR appears to be static in response to DLI relative to roots and leaves.

In addition to biomass accumulation and allocation, leaf morphology in terms of leaf area exhibited morphological plasticity in response to the DLI during root development (Fig. 2). For instance, we observed an increase and decrease in LAR and SLA for cuttings under a DLI of 2.0 and 13.4 mol·m⁻²·d⁻¹, respectively. The magnitude of the change between cuttings under the high and low DLI increased as DAT progressed. Similarly, Currey and Lopez (2012) reported a reduction of 37% and 29% in LAR and SLA, respectively, for new guinea impatiens ‘Magnum Salmon’ (shade-tolerant bedding plant) cuttings as DLI during root development increased by 13.1 mol·m⁻²·d⁻¹. This suggests that 7- to 14-d rooted cuttings respond similarly to more establish plants and may alter leaf traits as a result to acclimation to different light environments. For example, Nemali and van Iersel (2004b) reported that Begonia semperflorens-cultorum grown under a low DLI of 9.8 mol·m⁻²·d⁻¹ increased their LAR compared with plants grown under a DLI of 23.2 mol·m⁻²·d⁻¹ to capture a larger fraction of the available light. Additional acclimation responses to lower DLIs include a lower R:S and chlorophyll a/b ratio and increase in total chlorophyll content and SLA (Currey and Lopez, 2012; Nemali and van Iersel, 2004a).

A positive carbon balance was measured by the increase in biomass, regardless of magnitude, throughout root development across DLIs over the propagation period (Fig. 1A–I). This means that, regardless of the lighting treatment, gross daily photosynthesis was greater than daily carbon losses. This may be partially attributed to the fact that, even when ambient solar PPF was zero (i.e., predawn or postdusk), the PPF of supplemental light delivered for each DLI treatment (0600 to 2200 hr) was above the LCP for cuttings of each species under each DLI (Fig. 3). However, the differences in magnitude of increase of biomass accumulation across DLIs are likely attributed to P_n of cuttings under different DLIs. In a previous experiment, we grew new guinea impatiens cuttings under varying amounts of shade cloth with supplemental light from HPS lamps (Currey and Lopez, 2012). We collected survey measurements of P_n between 1130 and 1230 hr and found that the P_n of new guinea impatiens cuttings increased from ≈1‒2 μmol·m⁻²·s⁻¹ CO₂ to 6‒7 μmol·m⁻²·s⁻¹ CO₂ as the light intensity (ambient plus supplemental light) increased from 43.4 μmol·m⁻²·s⁻¹ PPF (low-light treatment) to 230.4 μmol·m⁻²·s⁻¹ PPF (high-light treatment), respectively (unpublished data). Although not measured in this experiment, we postulate that cuttings under the higher DLIs were exposed to a higher PPF throughout the photoperiod and, therefore, greater P_n occurred throughout the day for cuttings under high DLIs.

The P_gmax varied throughout propagation and across DLIs differently for species (Fig. 3D–F). For example, P_gmax of geranium and petunia decreased upon transfer into the low and medium DLIs, whereas P_gmax of cuttings under the highest DLI remained similar to the P_gmax of cuttings −1 DAT (callusing). The oldest leaves of geranium and petunia cuttings (present on the cutting at time of harvest from the stock plant) likely formed under a high-light environment, comparable to that of the high DLI treatment during root development. There are several potential explanations for the increasing P_gmax of impatiens upon transferring into higher DLIs. The leaves of cuttings may have acclimated to the darkness during the 2-d shipping or the low light during callusing. Alternatively, the initial leaves of impatiens cuttings may have been formed under a lower-light environment. It is not uncommon for instantaneous light levels in Central American stock plant facilities to approach ≈2000 μmol·m⁻²·s⁻¹ (C. Currey, personal observation). However, facilities where new guinea impatiens stock plants are grown are shaded in an effort to reduce light levels and unwanted flowering, as cuttings with a less reproductive meristem are desirable (R. Heins, personal communication). In addition, reducing the light intensity can diminish the potential for photodamage to leaves from high light intensities. Therefore, geranium and petunia (sun-loving species) stock plants are likely grown under high instantaneous light levels resulting in increased P_gmax, while new guinea stock plants may be grown under low instantaneous light levels with respect to P_n. Lopez (2007) studied the impact of DLI on stock plants and reported different responses among taxa. For example, cuttings of sun-loving species such as Thunbergia alata ‘Sunny Lemon Star’ exhibit increased root growth and percentage of rooted cuttings when harvested from stock plants grown under high DLIs of 12 to 15.1 mol·m⁻²·d⁻¹, whereas Jamesbrittenia grandiflora ‘Breeze Upright White’ and Verbena ×hybrida ‘Aztec Red Velvet’ showed a reduction in rooting and percentage rooted cuttings when grown under high DLIs of 12 to 15.1 mol·m^‒2·d⁻⁻¹ and 11.9 to 15 mol·m⁻²·d⁻¹, respectively. Cuttings of new guinea impatiens ‘Harmony Magenta’ had similar root growth regardless of stock plant DLI, though cutting harvest was greater for stock plants grown under higher DLIs. This presents a challenge to balance stock plant growth, maintenance, and productivity with subsequent rooting and photosynthetic potential during propagation for cuttings of vegetatively propagated taxa.

The dependency of cuttings on adventitious roots for photosynthesis may vary among plant species (Davis, 1988). For example, in our experiments, the P_gmax for geranium and petunia cuttings propagated under low DLIs with no or very few roots (−1 and 2 DAT) was equal or greater than cuttings with greater root mass (later in propagation), while P_gmax increased for new guinea impatiens cuttings, regardless of DLI, after root growth had begun (Figs. 1A–C and 3D–F). Klopotek et al. (2012) reported that from 3 to 13 d in propagation, P_n for petunia ‘Mitchell’ doubled from ≈2 to 4 μmol·m⁻²·s⁻¹ as the PPF increased from 80 to 150 μmol·m⁻²·s⁻¹ and appeared unaffected by the time or the presence of adventitious roots. Alternatively, Svenson et al. (1995) reported P_n of Euphorbia pulcherrima ‘Lilo’ and ‘Amy’ increased from 0.9‒2.2 to 7.4‒9.8 μmol·m⁻²·s⁻¹ and 1.0‒2.0 to 7.6‒9.1 μmol·m⁻²·s⁻¹, respectively, from before until after root emergence. This may be related to different species’ abilities to take up water through the stem or cuticle to maintain open stomata. From −1 to 5 DAT, the g_s and E of new guinea impatiens cuttings increased from 0.03‒0.04 to 0.08‒0.10 μmol·m⁻²·s⁻¹ and 0.6‒0.8 to 1.5‒2.0 mmol·m⁻²·s⁻¹, respectively (data not shown), coinciding with the development of measureable root mass (Fig. 1). Alternatively, the g_s and E of geranium cuttings were 0.16 μmol·m⁻²·s⁻¹ and 2.9 mmol·m⁻²·s⁻¹, respectively, by −1 DAT and were 0.07‒0.11 μmol·m⁻²·s⁻¹ and 1.3‒2.1 mmol·m⁻²·s⁻¹, respectively, from 2 DAT onward (data not shown), with no increase in g_s and E upon root emergence. The variation in g_s and E between cuttings of different species early in propagation with no root growth may be related to the stem or cuticular water uptake of taxa and ability to maintain turgid stomata and, thus, maintain a high P_n.

Our data pertaining to other parameters of photosynthetic responses with respect to light including R_d, α, LCP, and LSP are consistent with and complement existing data on the effects of light on leaf photosynthesis. For example, toward the latter half of the DLI treatments, we generally observed an increase in R_d, LCP, and LSP with increasing DLI, while α was unaffected. McDonald (2003) noted that it is common for sun and shade leaves of the same taxa to have similar α, irrespective of the light environment. Leaves adapted to different environments are able to maintain a balance in photosynthetic efficiency, because shade-adapted leaves have larger light-harvesting apparatuses and fewer photosynthetic reaction centers compared with sun leaves (Boardman, 1977). Furthermore, it is common for leaves with a high photosynthetic capacity (i.e., P_gmax and LSP) to have greater R_d and higher LCP to maintain activity associated with increased organelle activity (McDonald, 2003).

In addition to enhanced gas exchange under higher DLIs, we observed an increase in endogenous starch concentrations with increasing DLIs during root development. The increased starch concentration likely increases carbohydrates available for development and incorporation of root tissue and, therefore, contributes to the enhanced allocation of biomass into root growth. The cause for a reduction in carbohydrates for cuttings of new guinea impatiens, geranium, and petunia under the highest DLI at the end of 14 d from previous, higher concentrations is unclear (Fig. 3). The decline in shoot starch concentrations of petunia coincided with an increase in leaf growth (Fig. 1). This leads us to hypothesize that the cutting had entered a point in an episodic growth cycle where shoot growth began to increase and starch concentrations declined because of significant biomass allocation into the sink (leaf). However, sampling further beyond the 14-d treatment period would help elucidate patterns, if any, that appear during cutting propagation and help develop a better understanding of fluctuations in endogenous shoot carbohydrate levels for cuttings.

Although we did not transplant cuttings from different DLI treatments and grow them out to flower, previous studies have shown that cuttings rooted under higher DLIs may also flower more quickly following propagation (i.e., fewer days to flower) even though they appear to be similarly induced to flower (i.e., similar number of nodes below the flower). Hutchinson et al. (2012) hypothesized that the development of flower buds are a carbon sink and that enhancing endogenous carbohydrates as a result of increased light during propagation contributes to the developing sinks. Our data on the enhanced carbohydrate concentration of cuttings propagated under higher DLIs may further complement and strengthen this hypothesis.