Introduction

Strawberry (Fragaria × ananassa Duch.) is the most economically important small fruit crop in the Rosaceae family and can be consumed either fresh or processed (Castro & Lewers, 2016). Since its domestication, strawberry production has continuously increased. In 2019, its total world production was estimated to be 8.88 million tons (FAOstat). Strawberries contain several potential health-promoting phytonutrients, including phenolics, polyphenols, fibers, micronutrients, ellagic acid, ellagitannin, and vitamins (Aaby et al., 2012). Owing to their health-promoting benefits, ability to adapt to a wide range of environments, and income- rewarding properties, strawberries have attracted the interest of consumers, farmers, and researchers (Aaby et al., 2012). Cultivated octoploid strawberries are categorized into two main groups based on their flowering habits: the June-bearing type, also known as short-day or winter strawberries, and the ever-bearing type, also known as long-day or summer strawberries (Honjo et al., 2016). The June-bearing type is the most commonly used cultivar for commercial production in regions that experience winters, such as Korea, and its production is highly dependent on environmental conditions (interaction between temperature and photoperiod), especially for floral bud induction and initiation (Stewart & Folta, 2010). The plant transition from the vegetative to the reproductive phase is a physiologically and agriculturally important stage, as it is linked to the production potential of flowering crop species; however, proper timing and duration of this process are considered essential for fruit production (Perrotte et al., 2016). The decrease in temperature and photoperiod at the end of summer is accompanied by many physiological and morphological changes that lead to a low level of growth potential, preventing vegetative growth and development, which is characteristically indicated by a reduction in runner and petiole length (Robert et al., 1999). Furthermore, a change in biomass allocation and carbohydrate accumulation in different parts of the strawberry seedlings submitted to flower-inducing conditions are some of the physiological events reported to be related to flower initiation of June-bearing strawberries (Eshghi et al., 2007; Sønsteby et al., 2016).

No morphological or molecular markers that ordinary farmers and researchers can use to decide whether the seedlings in the nursery have initiated floral buds for transplanting to ensure successful and uniform flowering, thereby allowing yield prediction, have been identified. Given that this reduction in vegetative growth favors generative growth that occurs at the early beginning of the fall season owing to changes in environmental conditions, we hypothesized that close observation of the morphological and physiological changes that occur at this particular time can reveal the key phenotypes related to early flowering in young strawberry seedlings. Therefore, we investigated changes in plant morphology and physiology triggered by a reduction in environmental temperature and photoperiod during the seedling stage in this study.

Material and Methods

1. Plant material and handling

This study was conducted in a research greenhouse at the Korea Institute of Science and Technology (KIST), located in Gangneung-si, Gangwon-do, Republic of Korea. The seasonal-flowering (June-bearing cultivar) ‘Seolhyang’ strawberry mother plant materials were purchased from a professional farmer in Pyeongchang, Gangwon-do Province, and grown as mother plants in rectangular containers (80 × 35 × 20 cm) in a controlled greenhouse of KIST from September 2019 to February 2020 to produce runner tips. The containers were filled with commercial strawberry medium, planted with four plants each (20 cm spacing), and placed on a 1 m raised bed in the greenhouse. The greenhouse environmental conditions were kept at 24°C during the day, 25°C at night, and 24 h photoperiod to encourage rendering during the winter time. The day length was extended to 24 h using a 70 W incandescent lamp. Runner tips were harvested on February 14, 2020, and rooted in small pots (9 cm × 6 cm × 8 cm) using a commercial potting mix in a greenhouse. After they were uniformly rooted (two weeks), they were divided into two groups (treatments), each with ten replicates. One group of the seedlings remained in the greenhouse under non-flower-inducing conditions, characterized by continuous light exposure and day/night temperatures of approximately 24－25 °C. Another group was subjected to flower-inducing conditions by moving the seedlings every night (6:00 pm to 9:00 am) into a cooling chamber maintained at 15 °C nighttime temperature combined with a short-day photoperiod (approximately 8 h light / 16 h dark). This was done by moving plants to the cooled room every night (6:00 pm) and bringing them back to the greenhouse in the morning (9:00 am) for a period of 15 days. The 15-day treatment period was determined to capture early physiological and morphological responses related to floral induction. This period was selected based on the experimental objective to detect initial changes in plant development that precede visible flower formation. After 15 days of low-temperature and short-day treatments, all plants were kept under conventional management (supplying only water) for 2 months and then checked for flowering. The plants were manually irrigated daily, and no fertilizer was supplied throughout the experimental period. Data collection started 1 week after moving the plant materials to their respective conditions as follows: March 6, March 13, March 20, March 27, April 3, April 10, and April 17, 2020. Fifteen growth parameters, i.e., crown diameter, plant height, petiole length, leaf length, leaf width, leaf area, number of leaves, chlorophyll content, shoot fresh weight, shoot dry weight, number of main roots, root length, root fresh weight, root dry weight, and number of runners, were evaluated. On the day of data collection, plants were harvested in the early morning before the sun rose to minimize diurnal metabolic changes, removed from the pots, and the soil was washed off. After the fresh material measurements were taken, all plants were divided into roots, crowns, and leaves (lamina and petiole), bagged into open aluminum foils, and dried in an oven at 70°C for 3 days for dry matter analysis. The crown diameter was measured using a manual caliper, and the length and width characteristics were recorded using a measuring tape. Fresh and dry weights were measured using an electronic balance (Mettler Toledo ME 203; Shinsung Instrument Co., Ltd.), and chlorophyll content was determined using a hand-held SPAD 502 Plus Chlorophyll Meter (Konica-Minolta, Japan). Leaf area was measured using a green leaf area meter (Model GA-5, Tokyo Photoelectric Co., Ltd.).

2. Soluble sugar analysis

In addition to the growth traits, five additional replicates (two plants per replicate) were randomly sampled from both treatments for soluble sugar (sucrose, glucose, and fructose) analysis on the dates as growth data. The crown, leaves, and roots were separately freeze-dried at －80°C and ground using mortar and pestle.

Approximately 100 mg of dried plant material was weighed, transferred into an Eppendorf tube, and the soluble carbohydrates were extracted twice using 2 mL 80% ethanol and an ultrasonic bath (Model USC 200 TH, VWR, Leuven, Belgium) at 60°C for 30 min. The homogenates were centrifuged at 15,000 rpm for 3 min after each extraction and the resulting supernatants from the two extractions were combined. The ethanol was completely evaporated from the supernatant using a vacuum desiccator (Eppendorf AG 22331, Hamburg, Germany) at 60°C, then solubilized by adding 2 mL of water to the extracts and using the ultrasonic bath for 30 min at 60°C. The resulting extract was centrifuged at 15,000 rpm for 3 min and then filtered with a 0.45 μm GHP membrane filter (Millipore) before injecting 10 μL into a high performance liquid chromatograph (Agilent 1200 series of HPLC, Agilent Technologies, Waldbronn, Germany) fitted with a refractive index detector to separate and identify different soluble sugars. Sugars were separated using a specialized column for separating carbohydrates (Agilent Hi-Plex Ca USP L19, 4,0 * 250 nm, 8 μm; p/n PL1570-5810). The mobile phase was made of 100% water, supplied at the flow rate of 0.3 mL min^-1, and the column temperature was 80°C. The amount of sugar present in each sample was determined from calibration curves constructed using 100, 200, 500, 1000 or 2000 mg L^-1 standard solutions of sucrose, glucose and fructose that were injected into the column. The amounts of individual sugars in each sample were calculated using the peak areas and calibration curves.

Differences between treatments in plant growth parameters and soluble carbohydrates were analyzed using a paired t-testusing GraphPad Prism (GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com), and principal component analysis (PCA) was carried out using R software (R Core Team, 2021).

Results and Discussion

The results indicated noticeable morphological and physiological changes between the two groups of plants treated under different environmental conditions. Table 1 summarizes the observable morphological phenotypes recorded and analyzed at different time intervals. Most of the phenotypes examined in seedlings maintained under continuous light and high-temperature conditions quantitatively exceeded those of seedlings exposed to short-day and low-temperature conditions, particularly in shoot-related traits.

According to previous investigations, flower initiation starts after 21 days of short-day and low-temperature treatments, and macroscopic flowers form within 37－54 days (Jonkers, 1965; Ruan et al., 2011). We performed a t-test on data obtained 28 days after treatment to determine whether the observed differences were statistically significant. Most shoot traits, such as plant height, petiole length, leaf area, leaf count, shoot dry weight, and number of runners, were significantly higher (p < 0.05) in plants maintained under continuous light and high-temperature conditions compared to those exposed to short-day and low-temperature conditions. Conversely, crown diameter and root biomass were significantly enhanced in seedlings subjected to the short-day and low-temperature treatment.

Leaf features (length and width) and petiole length of the third fully expanded leaf (counted from the youngest leaf) were measured. The larger leaf length and width observed in plants subjected to short-day and low-temperature conditions may be explained by the more rapid production of smaller leaves in plants kept under continuous light and high temperature. A reduced number of leaves has also been reported as an indicator of plant phase change in Arabidopsis (Pouteau et al., 2006; Pouteau & Albertini, 2009). Reduced petiole length is commonly used to assess the decreased vegetative growth potential of strawberry plants under inductive environments (Robert et al., 1999; Sønsteby & Heide, 2006). Although not statistically significant, the petioles of leaves from seedlings exposed to short-day and low-temperature conditions were shorter than those of plants maintained under continuous light and high- temperature conditions. Notably, changes in leaf and stem characteristics are commonly associated with the transition from vegetative to reproductive development in higher plants (Poethig, 2013). This morphological distinction between the treatment groups was clearly evident 28 days after exposure, as visually represented in Fig. 1. Seedlings exposed to flower-inducing conditions exhibited compact growth with fewer and thicker leaves, whereas those in the control environment showed more elongated shoots and abundant runners.

Fig. 1.

Morphological appearance of strawberry seedlings grown under different environmental conditions 28 days after treatment. (A) Flower-inducing environment (short-day and low-temperature). (B) Non-flower-inducing environment (continuous light and high-temperature).

Overall, we observed differential biomass production and partitioning among plant organs depending on the environmental conditions provided (Fig. 3). Shoot biomass was significantly lower (p < 0.05) in seedlings exposed to short-day and low-temperature conditions compared to those maintained under continuous light and high-temperature conditions, whereas the opposite trend was observed for root biomass. As mentioned above, the increased shoot biomass in plants under continuous light and high-temperature conditions was attributable to the enhanced rate of leaf production and runner development (Fig. 2). Additionally, no fertilizers were applied during the experimental period. Consequently, while overall plant size was reduced, total biomass remained relatively high. These findings are consistent with previous results by Sønsteby et al. (2016) under controlled conditions. They found that strawberry seedlings grown under flower-inhibiting conditions (high temperature and extended photoperiod) exhibited greater dry matter accumulation and larger leaf area compared to those grown under flower-inducing conditions (low temperature and short photoperiod). Similar results were reported by Olsen et al. (1985) under field conditions. Furthermore, increased leaf area contributing to dry matter accumulation in high-temperature environments has also been documented in crops such as rice, tomato, and G. officinalis (Hussey, 1963; Patterson, 1993).

Fig. 2.

Biplot representation of the principal component analysis (PCA) results illustrating the morphological and physiological characteristics of ‘Seolhyang’ strawberry seedlings under control and treatment conditions. The PCA biplot shows clear variance between the two groups based on parameters such as plant height (PH), petiole length (PL), crown diameter (CD), leaf number (LN), root number (RN), root length (RL), leaf length (LL), leaf width (LW), width-to-length ratio (WLR), chlorophyll concentration (CC), leaf area (LA), shoot fresh weight (SFW), root fresh weight (RFW), number of runners (RUN), shoot dry weight (SDW), root dry weight (RDW), leaf dry weight (LDW), and crown dry weight (CDW). The first two principal components explain 31.8% (PC1) and 17.0% (PC2) of the total variance. Each point represents a unique plant sample from the respective treatment group.

Fig. 3.

Time-course analysis of dry matter accumulation in ‘Seolhyang’ strawberry seedlings under flower-inducing and non-inducing environments. The graphs display partitioned dry matter accumulation in (left) leaves, (center) crowns, and (right) roots over 49 days following treatment. Each curve represents the mean value (n = 10), with error bars indicating the standard error of the mean. Treatment conditions conducive to flowering are compared against non-inducing control conditions.

Fig. 4 illustrates the chlorophyll content, measured as SPAD values, in strawberry seedlings over a 49-day period under the control and flower-inducing treatment conditions. The SPAD values for both conditions initially trended upward, indicative of increased chlorophyll concentration during early plant development.

Fig. 4.

Changes in chlorophyll content (SPAD values) of ‘Seolhyang’ strawberry seedlings over 49 days following exposure to flower- inducing and non-inducing conditions. Values represent means ± standard deviation (n = 10).

The chlorophyll content in the control group peaked approximately 20 days after treatment and then plateaued, with a slight decrease observed after day 40. The treatment group followed a similar initial trend but with a subdued peak, suggesting less vigorous vegetative growth. The subsequent decline in the SPAD values was more pronounced in the treatment group. This suggested that seedlings under flower-inducing conditions may have shifted their energy allocation to reproductive development, resulting in a reduced demand for chlorophyll.

The consistently higher SPAD values in the control group implied that a non-flowering environment fostered greater chlorophyll retention, likely because of vegetative growth focus. The observed late-stage decline in the control group could be attributed to nutrient depletion in the soil, as the initially vigorous vegetative growth would rapidly deplete resources without replenishment. Conversely, the treated seedlings exhibited moderate use of nutrients, aligned with their slower growth and generative phase transition, leading to a more gradual decline in chlorophyll content.

The PCA biplot (Fig. 2) offers a comprehensive overview of the variance in morphological and physiological traits of ‘Seolhyang’ strawberry seedlings under control and treatment conditions. The biplot facilitated the comparison of the two distinct environmental effects on the seedlings by projecting multidimensional data onto the principal components Dim1 and Dim2, which together explained 50% of the total variance. Dim1, which accounted for 34.5% of the variance, appeared to be strongly correlated with root fresh weight (RFW), root dry weight (RDW), and crown diameter (CD), which were more prominent in the treatment group. This suggested that flower-inducing treatments may promote root development and crown expansion, possibly as part of the shift towards reproductive growth. Conversely, the control group, which was associated with higher Dim2 values (explaining 15.5% of the variance), was characterized by traits related to vegetative growth, such as leaf number (LN), leaf area (LA), shoot fresh weight (SFW), and number of runners (RUN). These traits indicate a more robust vegetative phase, potentially because of the absence of flower-inducing stress. The distinction between the groups was evident, with treatment conditions fostering traits associated with flowering readiness, whereas the control conditions supported vigorous vegetative growth. These observations were similar to those reported by Masaru et al. (2016), where low temperatures enhanced root activity and development as well as leaf chlorophyll concentration. This parallels our observations in Fig. 2, which showed that strawberry seedlings under cooler flower-inducing conditions developed increased root mass, leaf chlorophyll concentration, and crown diameter. These features may indicate a transition from strawberry growth to reproductive growth. Additionally, an association between larger crown diameters and significantly earlier flower initiation and yield has been previously reported (Fridiaa et al., 2016; Torres-Quezada et al., 2015).

1. Soluble sugar partitioning

The results, as depicted in Fig. 5, elucidated the temporal allocation of soluble sugars (sucrose, glucose, and fructose) within various tissues of strawberry seedlings under different environmental conditions. During early development (14 and 28 days after treatment), glucose predominated in the leaf and root tissues under both non-flowering and flower-inducing conditions, with the exception of the crown tissues at 14 days, where the sucrose concentration was higher in the non-flowering environment.

Fig. 5.

Time-course changes in the concentrations of soluble sugars in strawberry seedlings grown under control and flower-inducing conditions. Sucrose (A, D, G), glucose (B, E, H), and fructose (C, F, I) levels were measured in the leaves (A–C), crowns (D–F), and roots (G–I), respectively. Bars represent the standard deviation (SD) of ten biological replicates.

A shift was observed 48 days after treatment, with sucrose becoming the leading sugar across all plant tissues, regardless of the environmental conditions. This was especially pronounced in the crown tissues of seedlings under flower- inducing conditions, where sucrose levels remained consistently elevated, a statistically significant finding as indicated by the t-test results (P < 0.05).

Seedlings in non-flowering conditions demonstrate higher concentrations of glucose and fructose across all tissues, resonating with the findings of Sønsteby et al. (2016), who reported a predominant accumulation of sugars in the leaves and identified glucose as the most abundant under non- flowering conditions. However, by 48 days, contrary to the observations of Sønsteby et al. (2016), sucrose emerged as the most abundant sugar in both environmental conditions, which may be attributed to differences in environmental conditions and the longer duration of data collection in the present study.

The significant increase in sucrose within the crown tissues of seedlings under flower-promoting conditions at 48 d aligns with the argument by Eshghi et al. (2007) that, at the onset of flower bud initiation, shoot tips become a strong sink, actively utilizing carbohydrates rather than storing them, as suggested by Peng and Iwahori (1994). Concurrently, induced plant shoot tips may compete with the roots for carbohydrate resources, as further indicated by Eshghi et al. (2007). After 28 days of treatment, the sucrose concentration in induced plants was double that in non-induced seedlings, potentially signaling the initiation of flowering. This is supported by the theory that sucrose accumulation in reserve organs triggers gibberellin synthesis, facilitating the transition from vegetative growth to flowering (Kurokura et al., 2013).

In conclusion, our study elucidated the significant impact of environmental conditions on the morphological and physiological development of ‘Seolhyang’ strawberry seedlings. Flower-inducing treatments, characterized by cooler temperatures and shorter daylight hours, promoted root development and crown expansion, potentially indicating a shift toward reproductive growth. In contrast, seedlings grown under control conditions exhibited more vigorous vegetative growth with increased leaf production and larger leaf areas. These findings align with existing literature, confirming that strategic environmental manipulation can effectively influence the balance between the vegetative and reproductive phases of strawberry plants, with implications for optimizing yield and quality in agricultural practices.