Watermelon Seedling Quality, Growth, and Development as Affected by Grafting and Chilling Exposure During Simulated Transportation

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Authors:
John M. ErtleDepartment of Horticulture and Crop Science, The Ohio State University, Columbus, OH 43210

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Chieri KubotaDepartment of Horticulture and Crop Science, The Ohio State University, Columbus, OH 43210

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Grafted watermelon plants available in the United States are typically transported for a long distance from a specialized nursery to the production field. To investigate the effects of chilling stress during transportation on the early plant growth and development, grafted and nongrafted ‘Tri-X-313’ seedless watermelon (Citrullus lanatus) seedlings were subjected to low-temperature treatments applied over a 72-hour period. The first experiment exposed grafted and nongrafted seedlings to 0, 6, 12, 24, or 48 hours of 1 °C chilling, and then were moved to a 12 °C growth chamber for the remainder of the chilling treatment period. The second experiment exposed nongrafted seedlings to seven different combinations of chilling duration (0, 24, 32, 41, 44, or 48 hours) to create varied chilling degree hours (CDH) at different temperatures (between −0.4 °C and 1.2 °C). After 72 hours, seedlings were transplanted in pots filled with a commercial substrate in a greenhouse to evaluate the early plant growth and floral development. Each experiment had two repeats (spring and summer) with a randomized complete block design (n = 10). Although greater exposure to chilling negatively affected visual quality and photosynthetic capacity [measured by chlorophyll fluorescence parameter, variable fluorescence/maximum fluorescence (Fv/Fm)] in both repeats, delay in flowering after transplanting was significant in spring only and increased with increasing CDH (up to 6 days with 48 hours of 1 °C exposure). Grafting was found to mitigate the degree of flowering delay when the same chilling exposure was applied. When chilling temperatures were varied, visual damage of leaves, decrease in Fv/Fm, and delays in female flower development were best correlated with CDH at a base temperature of 15 °C, 3 °C, and 4 °C, respectively. Our experiments and further analyses with available literature data suggest that 50 to 70 CDH4 [CDH with base temperature (BT) = 4 °C] seems to be a critical threshold to cause significant delay in female flower development (3.5 days for grafted and 1.3 days for nongrafted plants). Therefore, if temperatures lower than 4 °C are expected during transportation of seedlings, we suggest mitigation measures be taken so that CDH4 do not reach greater than 50 degree hours.

Abstract

Grafted watermelon plants available in the United States are typically transported for a long distance from a specialized nursery to the production field. To investigate the effects of chilling stress during transportation on the early plant growth and development, grafted and nongrafted ‘Tri-X-313’ seedless watermelon (Citrullus lanatus) seedlings were subjected to low-temperature treatments applied over a 72-hour period. The first experiment exposed grafted and nongrafted seedlings to 0, 6, 12, 24, or 48 hours of 1 °C chilling, and then were moved to a 12 °C growth chamber for the remainder of the chilling treatment period. The second experiment exposed nongrafted seedlings to seven different combinations of chilling duration (0, 24, 32, 41, 44, or 48 hours) to create varied chilling degree hours (CDH) at different temperatures (between −0.4 °C and 1.2 °C). After 72 hours, seedlings were transplanted in pots filled with a commercial substrate in a greenhouse to evaluate the early plant growth and floral development. Each experiment had two repeats (spring and summer) with a randomized complete block design (n = 10). Although greater exposure to chilling negatively affected visual quality and photosynthetic capacity [measured by chlorophyll fluorescence parameter, variable fluorescence/maximum fluorescence (Fv/Fm)] in both repeats, delay in flowering after transplanting was significant in spring only and increased with increasing CDH (up to 6 days with 48 hours of 1 °C exposure). Grafting was found to mitigate the degree of flowering delay when the same chilling exposure was applied. When chilling temperatures were varied, visual damage of leaves, decrease in Fv/Fm, and delays in female flower development were best correlated with CDH at a base temperature of 15 °C, 3 °C, and 4 °C, respectively. Our experiments and further analyses with available literature data suggest that 50 to 70 CDH4 [CDH with base temperature (BT) = 4 °C] seems to be a critical threshold to cause significant delay in female flower development (3.5 days for grafted and 1.3 days for nongrafted plants). Therefore, if temperatures lower than 4 °C are expected during transportation of seedlings, we suggest mitigation measures be taken so that CDH4 do not reach greater than 50 degree hours.

Grafting of fruiting vegetable crops such as watermelon (Citrullus lanatus) is widely adopted to reduce the incidence of soil-borne disease in many countries (Davis et al., 2008; Lee et al., 2010). Grafted plants also provide increased yields, improved fruit quality, or improved tolerance to abiotic stress (Lee et al., 2010). Grafting in North America was first introduced in greenhouse tomato (Solanum lycopersicum) in the late 1990s (Kubota, 2016), and more recently has been introduced for tomato in high tunnels and watermelon in the open field. As of 2019, watermelon accounts for ≈67% of all grafted seedlings (estimated ≈5 million plants) produced in the United States (Ertle and Kubota, 2020). Watermelon transplanting is typically targeted for 2 weeks after the final frost of a given climate region (Elwakil et al., 2017) and can be as early as February in Arizona, Florida, and the southern desert of California. As the number of grafting nurseries is still limited in the United States, grafted watermelon seedlings are often shipped long distances (up to 5 d) and may be exposed to suboptimal temperatures during transportation.

Transportation in unfavorable conditions is known to reduce the quality of live plants. Bulk transport of live plants is a major industry, as 80% of floriculture products (e.g., live plants, cut flowers) sold in the United States are imported from other countries (Steiner, 2018). The most common distribution method in the United States is by trailer, and guidelines (e.g., USDA ARS, 2016) are available for transporting flowers, ornamental potted plants, and bedding plants. In contrast, limited information is available on the optimum conditions for transporting vegetable seedlings, as they are historically produced in local or regional nurseries. Because of the limited number of grafting nurseries in North America, long-distance transportation is common, and millions of grafted seedlings travel for many days at a time in various seasons. Cantliffe (1993) summarized that transplant survival, posttransplanting quality, and yield of various vegetable crops were negatively affected by high-density packaging, low temperatures, transplant age, and exposure to darkness during transportation. To our knowledge, no information on transporting watermelon transplants is available despite watermelon generally being the most sensitive to chilling stress among common grafted species (Kubota et al., 2017). Therefore, better understanding on potential adverse effects of chilling exposure is crucial.

Plants are generally known to exhibit a quantitative response to low temperature and the duration of exposure. The lower the temperature becomes and/or the longer the exposure continues, damage to plants increases. However, when the temperature is above a specific threshold temperature, plants can tolerate with little to no cumulative damage. Therefore, chilling stress can be expressed using CDHs relative to the threshold (BT). Thermal time calculations combine temperature and time into a single number, often to assess plant development. In this case, CDH is a rearrangement of thermal time to assess responses to chilling stress. Cumulative chilling response is used to understand dormancy, whereas CDH (below a threshold BT) determines the minimum chilling requirement for breaking winter dormancy of various perennial species. For example, peach (Prunus persica) requires a minimum of 200 CDHs with a BT of 6 °C to emerge from dormancy (Erez et al., 1990). Sparks (1993) exposed mature pecan (Carya illinoinensis) trees to different durations (0–800 h) of chilling at temperatures between −1.7 and 4.4 °C to determine budbreak requirements, finding that CDH computed with a BT of 3.9 °C was the most accurate, and increasing CDH led to earlier flowering. Breaking dormancy of strawberry (Fragaria ×ananassa) plants also depends on CDH. Strawberry nurseries chill plants to accumulate CDH in an optimal range between 2.2 and 8.9 °C and initiate more rapid flower development once the plants are in the growing environment (Tanino and Wang, 2008).

Although the concept of CDH with species-specific BT has not been applied to acute chilling stress and subsequent injury of vegetable seedlings, previous studies showed facultative chilling stress responses to temperature and relatively short exposure time. For example, Kozik and Wehner (2014) found that 14 cultivars of watermelon seedlings showed increasing degree of chilling injury (CI) with increasing hours (12–36 h) of exposure to 4 °C. They also found that the exposure to 2 °C for 24 h, a shorter duration at a lower temperature, produced similar seedling damage at 4 °C for 36 h, indicating a reciprocity characteristic determining the same cumulative chilling response. Korkmaz and Dufault (2001) showed that watermelon plants reportedly reduced posttransplanting shoot and root fresh weight, leaf area, and vine length at a greater extent with increasing duration of exposure (9–81 h) to 2 °C during seedling stage. They also reported that days to first male and female flower emergence and days to fruit set increased linearly with increasing chilling duration. However, earlier studies showed that exposure to 3 °C low temperature up to 48 h (Ertle et al., 2021) or 12 °C up to 14 d (Spalholz and Kubota, 2017) did not adversely affect watermelon plant growth and development after transplanting, possibly because these temperatures were below BT for causing CI on watermelon. Knowing specific BT and a threshold CDH causing physiological and developmental damage would help nurseries and growers avoid undesirable chilling stress during long-distance transportation in winter. As grafting to a chilling-tolerant rootstock is known to mitigate the stress to chilling-sensitive scion plants (Li et al., 2008; Liu et al., 2004; Darré et al., 2021; Spalholz and Kubota, 2017), CI caused by acute temperature stress could be mitigated by chilling-tolerant rootstock such as interspecific squash (Cucurbita maxima × Cucurbita moschata).

In the present study, we hypothesized that 1) facultative responses to acute chilling stress exist for watermelon seedlings and the responses can be modeled using CDH below specific BT, and 2) a chilling-tolerant squash rootstock can improve the tolerance of watermelon plants to the acute chilling stress. To this end, we investigated effects of grafting and varied durations (0–48 h) of acute chilling (1 °C) (Expt. 1) and varied combinations of chilling temperatures (−0.4 to 1.2 °C) and durations of exposure (0–48 h) (Expt. 2) on the transplant quality and posttransplanting (into the greenhouse) growth and development of watermelon plants. In Expt. 2, we aimed to find specific BT and minimum CDH that could model the chilling-related plant responses. The chilling temperatures and durations examined in this study were selected based on our previous studies (Ertle et al., 2021).

Materials and Methods

Plant materials and early growing conditions.

Two different experiments (Expts. 1 and 2) were conducted, each repeated twice, once in spring and once in summer. Grafted and nongrafted seedlings were used in Expt. 1, and only nongrafted seedlings were used in Expt. 2. Watermelon cultivar Tri-X 313 (Syngenta Seeds, Boise, ID) and interspecific hybrid squash (Cucurbita maxima × Cucurbita moschata) ‘Strong Tosa’ (Syngenta Seeds, Wilmington, DE) rootstock were used. Seeds were germinated in 98-count plug trays filled with Fafard #2 substrate (Sungro Horticulture, Agawam, MA) and placed in a dark growth chamber at 28 °C for 2 days. Seedlings were then moved to a glass greenhouse (92.9 m2 floor area; Columbus, OH) set at 19/24 °C night/day air temperatures. The greenhouse temperature was controlled by pad-and-fan evaporative cooling and hot water radiant heating along the perimeter of the greenhouse. Heating and cooling setpoints were 23 ± 1 °C during daytime, and 19 ± 1 °C during nighttime. Supplemental overhead lighting in the greenhouse was provided for a maximum of 15.5 h·d−1 × 1000 W metal halide lamps (PARsource, Petaluma, CA) set to turn off 30 min before dusk. Supplemental lighting was activated when there was less than 350 W·m−2 of solar radiation (measured by an outdoor weather station on top of the greenhouse) for 10 min and turned off after 15 min of light above 400 W·m−2. If activated for the full 15.5 h, supplemental light provided 3.6 ± 0.6 mol·m−2·d−1 daily light integral (DLI). Seedlings were subirrigated with water (up to 14th day after seeding) and then with nutrient solution (half strength University of Arizona multicrop growth formula) containing (mg·L−1): 95 NO3-N, 24 P, 175 K, 100 Ca, 30 Mg, 58 S, 49 Cl, and micronutrients.

Grafted plants for Expt.1 were seeded 7 d earlier than nongrafted watermelon plants to account for the 7-d healing period. Grafting was conducted using a single cotyledon grafting method, and then plants were placed for 7 d inside a sealed, clear container (47-L volume, 45.7 × 66.0 × 22.9 cm) (Rubbermaid, Atlanta, GA) in a growth chamber (Conviron, Winnipeg, MB, Canada). Continuous lighting at 120 µmol·m−2·s−1 of photosynthetic photon flux density (PPFD) (measured on top of the container) provided by white fluorescent lamps (F72T12-CW-HO, 85 W; GE, Boston, MA) was used for the 7-d healing except for the first 24 h (in darkness). The chambers targeted 28 °C air temperature inside the healing container. Relative humidity inside the healing container was 100% during the first 5 d and gradually lowered over the final 2 d by opening the container lid a small amount at a time. After 7 d in healing, the seedlings were removed from the container and returned to the glass greenhouse and well-watered, followed by 7 d of hardening with reduced watering before the low-temperature treatments, together with nongrafted seedlings.

Low-temperature treatments.

Low-temperature treatments were applied over a 72-h period in both experiments to simulate chilling stress during transportation. Expt. 1 exposed grafted and nongrafted seedlings to 0, 6, 12, 24, or 48 h of 1 °C chilling, and then were moved to a 12 °C growth chamber for the remainder of the 72-h treatment period. Expt. 2 exposed nongrafted seedlings to 0, 24, 32, 41, 44, or 48 h of selected chilling temperatures in a range between −0.4 and 1.2 °C (Table 1) before transferring to 12 °C for the rest of the 72-h period. These low-temperature treatments were performed inside 31-L or 47-L containers (46 × 31 × 23 cm; or 46 × 66 × 23 cm; Rubbermaid) kept in dark temperature-controlled chambers. Different container sizes were used because of space limitations within the chilling chambers. Respectively, 15 or 30 plants were placed into small or large treatment containers. For temperature control in Expt. 1, two identical reach-in growth chambers were used (Model E15; Conviron). For Expt. 2, two chambers were designated for treatments above 0 °C (Sheldon Manufacturing Inc., Cornelius, OR), and those below 0 °C (ThermoFisher Scientific, Waltham, MA), and one chamber was designated for 12 °C (Model E15; Conviron). The locations of treatment containers inside each chamber were randomized.

Table 1.

Chilling temperatures, duration of acute chilling, and duration of 12 °C storage in Expt. 2 (spring). Trt = Treatment.

Table 1.

Posttransplanting growing conditions.

Following the 72-h low-temperature treatments, seedlings were placed on the greenhouse bench for 2 d before transplanting. Ten randomly selected seedlings from each treatment were transplanted into 3.8-L black plastic pots (diameter at rim = 16.5 cm; one plant per pot) in the greenhouse). The substrate used was BM-1 All-Purpose mix (Berger, Quebec, Canada). The plants were arranged into five double rows (90 cm apart) with 10 plants per row and plants spaced 30 cm apart in-row (3.3 plants/m2), and rows were oriented North/South. As plant growth progressed, plants were individually strung up by vine clips and horticultural twine to a wire 2.2 m above each row of pots to support vines upright. These practices were chosen for effectively observing differences in early plant growth and flower development among plants grown at this high planting density.

All plants were drip-irrigated with the nutrient solution mentioned earlier. Irrigation run time was 3 min, distributing 110 mL per run per plant with up to six runs per day at the height of vegetative growth. The number of irrigation runs was adjusted according to the percent drainage measured from the pot and targeted 30% drainage of the daily irrigation. Drip solution pH and electrical conductivity (EC) were adjusted to 5.5 to 6.5 and 1.3 to 1.4 dS·m−1, respectively, to achieve efflux drainage between 6.0 and 6.5 pH and under 1.7 dS·m−1 EC.

Data collection and analyses.

Seedlings were assessed for visual quality of all true leaves (>1 cm2) 1 h before the start of the 72-h low-temperature treatment, and 24 h after returning to the greenhouse. Visual assessments were conducted by the same person in the same laboratory condition using the Horsfall-Barratt scale (H-B, Table 2) based on approximate percentage leaf area with chlorosis and/or necrosis. For analysis, the H-B scores were converted to interval midpoints (Table 2), and then used to quantify the difference in damage observed over the time between immediately before and 24 h after treatment (ΔMP) for individual plants. This midpoint transformation is commonly used for H-B scores, as it reflects the variable ranges of area damaged (Bock et al., 2010).

Table 2.

Horsfall-Barratt scale values, ranges, and interval midpoints.z

Table 2.

Before and immediately after the 72-h low-temperature treatments (except for the first repeat of Expt. 1, for which this analysis was not performed), chlorophyll fluorescence was evaluated on the newest true leaf with an area larger than 2 cm2 using a Ciras 3 Portable Photosynthesis System (PP Systems, Amesbury, MA) with an actinic light (Red/Green/Blue at 38%/37%/25%) at a PPFD of 9000 µmol·m−2·s−1. Eight plants before, or six plants following the end of the 72-h low-temperature treatments, were randomly selected per treatment and dark-adapted for 1 h at 28 °C before measuring chlorophyll fluorescence. Chlorophyll fluorescence induction parameter Fv/Fm was selected, as it reflects the maximum potential quantum efficiency of photosystem II (Maxwell and Johnson, 2000) and therefore the ideal measurement for assessing leaf photosynthetic capacity as affected by the chilling stress.

After transplanting, the plants were assessed for vegetative growth weekly. Flower anthesis and nodal position of first male and female flowers were recorded daily. Vegetative growth measurements included leaf count, primary vine length, and internodal lengths. These measurements were then converted to a per-day increase.

Environmental conditions were monitored throughout the experiment. Specifically, air temperature in the greenhouse was monitored using calibrated T-type thermocouples (gauge: 36) spaced centrally above the seedling trays or in each experimental block. Additional air temperature and relative humidity (converted to air-based vapor pressure deficit, VPD) were measured using a fan-aspirated HMP60 probe (Vaisala; Vantaa, Helsinki, Finland) inside a radiation shield hung at a central location in the greenhouse. PPFD of natural light was monitored in the greenhouse attic using an LI-90R quantum sensor (LI-COR, Lincoln, NE). Air temperatures in chambers and containers used for healing and low-temperature treatments were monitored by calibrated T-type thermocouples (gauge: 24) (two thermocouples per chamber). All sensor readings were recorded with a CR10X or CR1000 datalogger (Campbell Scientific, Logan, UT). In addition, HOBO U23 dataloggers (Onset Computer Co, Bourne, MA) were placed inside all healing containers in the growth chamber for monitoring relative humidity, later converted to VPD. The same loggers were also placed in one container in each chamber during the chilling treatments.

Cumulative CDH in the present study was computed using the following equation:
CDH=Σi=1n[(BTTi)×Δt],
where BT is a selected base temperature (°C), T is a single temperature point of a chilling treatment measured inside the container (°C), and Δt is the sampling interval (h) of temperatures (Δt = 0.083 when TiBT, otherwise Δt = 0). As BT for inducing chilling stress on watermelon seedlings is unknown, we computed CDH with various BT in a range of 3 to 15°C with one degree increment, to determine a BT that shows the best correlation between CDH and the plant response of interest (e.g., days to the first female flowering).

Experimental design and statistical analysis.

For Expt. 1, the spring and summer repeats were conducted for 22 Feb. to 20 May 2019 and 10 May to 21 July 2019, respectively. For Expt. 2, the summer and spring repeats were for 9 July to 3 Sep. 2019 and 13 Dec. 2019 to 14 Mar. 2020, respectively. For pretransplanting evaluations (visual quality and Fv/Fm), the container used for low-temperature treatments was considered the experimental unit, with plants inside the container considered sampling units. The experimental model included the container [random; degrees of freedom (DF) = 4 in Expt. 1 and DF = 15 in Expt. 2], the treatment (fixed; DF = 9 in Expt. 1 and DF = 6 in Expt. 2), and the residual error.

For the posttransplanting measurements of vegetative growth and floral development, each plant was considered its own experimental unit, as no container effect was found when analyzing our results from the seedling chilling portion of the experiment. Plants were arranged in a randomized complete block design (RCBD) that divided the five double rows into North and South sections (10 blocks with 10 plants per block, 100 total plants) in the greenhouse. Expt. 1 allowed for two plants (one grafted and one nongrafted plant) of each of the five chilling treatments to be placed in each block, and Expt. 2 contained one to two nongrafted plants of each chilling treatment in each block. Thus, the RCBD blocking factor (10 blocks in the greenhouse) was considered as the only random effect to explain variation within the posttransplanting portion of the experiment. The experimental model included the block (random; DF = 9), the treatment (fixed; DF = 9 in Expt. 1 and DF = 6 in Expt. 2), and the residual error.

All statistical analyses were conducted using two statistical software programs (JMP Pro version 14.0.0; SAS Institute Inc., Cary, NC; RStudio version 1.4.1106; PBC, Boston, MA). Linear or nonlinear regressions were made to correlate CDH and specific plant responses (e.g., days to flower) using the JMP Nonlinear Model. A Wald’s test was used to determine the effects of blocking on the model. A Tukey’s honestly significant difference test and a pairwise t test were used to separate means (P ≤ 0.05).

Results and Discussion

Growth chamber environment.

Temperature differences measured within the containers during germination and graft healing were negligible between containers in the same growth chambers (data not shown). Graft healing temperatures within Expt. 1 averaged 26.9 ± 1.1 °C and 27.7 ± 1.0 °C for the spring and summer repeats, respectively. VPD was 0.08 ± 0.03 and 0.09 ± 0.03 kPa. Light intensity at the top of the containers were 122.0 ± 2.0 µmol·m−2·s−1 in both repeats.

During the low-temperature treatments, once containers were placed in the growth chambers, the internal temperature of the container matched the chamber temperature within 120 min and varied within less than 0.2 °C of the setpoints in Expt. 1. However, temperatures achieved within the containers in Expt. 2 varied greatly from the original setpoints we selected because of growth chamber variation. Therefore, our analyses and CDH calculations were based on the temperatures recorded within the containers of each treatment group (as shown in Table 1) instead of setpoint temperatures. VPD within each container used in the experiments was less than 0.1 kPa (data not shown) for both experiments.

Greenhouse environment.

Table 3 summarizes the environmental conditions of the greenhouse. Average solar DLI during the seedling growth stages ranged from ≈5.6 to 24.4 mol·m−2·d−1, and 8.1 to 23.7 mol·m−2·d−1 during the posttransplanting stages. Air temperatures did not vary greatly from setpoints conducted during spring months when the greenhouse received lower solar DLI. However, this was not the case in the summer months, which experienced consistently higher temperatures than the setpoints.

Table 3.

Greenhouse environmental conditions of the seedling and posttransplanting environment, means ± standard errors.

Table 3.

Environmental conditions in the greenhouse had a strong impact on the posttransplanting growth of plants following acute chilling. Specifically, none of the posttransplanting variables measured in the repeats conducted in summer months were significantly affected by the low-temperature treatments (data not shown), whereas the other repeats conducted in spring months showed significant facultative responses to chilling treatments (as described later). A possible reason could be a more advanced reproductive development of seedlings relative to the timing of chilling stress when seedlings were grown under higher temperatures in summer as further discussed in a later section. Another reason may be that the higher light intensities, longer daylengths, and higher day and night temperatures during posttransplanting growth and development may enhance the growth rate enough to mitigate any observable delays in flower anthesis. Summer temperature and light conditions masked the effects of acute chilling stress for both experiments. This aspect of light and temperature effects on crop development needs to be further investigated. Thus, results of the spring repeats are primarily discussed.

Expt. 1: Effects of 1 °C chilling durations on grafted and nongrafted seedlings.

The summer repeat of Expt. 1 did not show significant effects of chilling treatments on posttransplanting plant growth, and developmental parameters (data not shown). Therefore, the following data analysis and discussion mainly focuses on the results of the spring repeat of Expt. 1.

Seedling visual quality.

Positive ΔMPs were recorded in both repeats in this experiment, indicating the visual degradation of visual quality as affected by chilling at 1 °C. The cause of degradation was due to leaf chlorosis (yellowing), necrosis, and wilting, and a significant positive linear correlation was observed between ΔMP and duration of 1 °C exposure (Fig. 1A). When chilled, low temperatures can disrupt typical physiological functions by disrupting membrane proteins and lipids that play critical roles in plant structure and processes (Lyons, 1973). Activation energy for basic metabolic processes (e.g., light harvesting in the photosystem) increases as functional proteins decrease in plant cells (Janmohammadi, 2012), and ion leakage can lead to the accumulation of metabolites such as pyruvate, acetaldehyde, and ethanol, which can damage cells when concentrated in high amounts (Lyons, 1973). As a result, cells and tissues are not able to carry out normal function and can result in damage that leads to leaf yellowing, wilting, or necrosis, as we saw in this experiment. Because of the disruptive nature of chilling, plant tissues have reduced capacity to deal with environmental stress and may suffer increased damage posttransplant due to interference with normal mechanisms used to decrease oxidative stress (Allen and Ort, 2001).

Fig. 1.
Fig. 1.

(A) Percent change in leaf tissue damage (ΔMP) of grafted and nongrafted seedlings in Expt. 1 (spring), 24 h after the end of chilling as affected by the duration of exposure to 1 °C. Means ± standard errors (n = 5 treatment containers). (B) Chlorophyll fluorescence parameter variable fluorescence/maximum fluorescence (Fv/Fm) as affected by duration of exposure to 1 °C in Expt. 1 (summer). Data of grafted and nongrafted plants were combined, as there was no significant effect of grafting on Fv/Fm. Means ± standard errors (n = 4 treatment containers). (C) Days from transplant to first female flower anthesis for grafted and nongrafted plants as affected by durations of exposure to 1 °C in Expt. 1 (spring). Means ± standard errors (n = 10 blocks).

Citation: HortScience 57, 8; 10.21273/HORTSCI16557-22

In this experiment, ΔMPs for grafted plants were greater than those for nongrafted plants when the durations of 1 °C exposure exceeds 10 h (Fig. 1A). For example, the seedlings chilled for 48 h reached 42.7% ± 5.7% and 19.8% ± 3.2% surface damage for grafted and nongrafted seedlings, respectively. This trend was similar between both repeats and was also observed in our earlier similar study with 3 °C chilling (Ertle et al., 2021). Likely, this was due to the additional stress of graft healing before the chilling treatments that reduced seedling tolerance to chilling temperatures.

Chlorophyll fluorescence.

Chlorophyll fluorescence Fv/Fm before chilling was 0.72 ± 0.01 and did not differ between grafted and nongrafted plants. The Fv/Fm measured immediately after the 72-h low-temperature treatments decreased with increasing the duration of 1 °C chilling regardless of grafting (Fig. 1B). Seedlings with 48 h of 1 °C chilling had the lowest Fv/Fm, 11% lower than the nonchilled control treatment (72 h of 12 °C storage without 1 °C chilling). These significant reductions in Fv/Fm indicate a reduced photosynthetic capacity of the tissue, and is measurable before visually detectable symptoms (e.g., chlorosis, necrosis) emerge.

Chlorophyll fluorescence has been used widely for assessing physiological injuries caused by abiotic stress on plants, including low temperature. Hou et al. (2016) found that Fv/Fm of watermelon seedlings declined from 0.82 to 0.75 after 3 d under a 12/10 °C day/night temperature and an irradiance of 250 µmol·m−2·s−1 in a growth chamber. They also showed that reducing light to 80 µmol·m−2·s−1 under the same temperature regimen allowed the seedlings to maintain an Fv/Fm of 0.77. Ding et al. (2011) showed that grafted or nongrafted watermelon seedlings stored in darkness at 15 °C for 6 d reduced Fv/Fm from 0.8 prestorage to 0.77 to 0.78 poststorage. However, these levels of reduction in Fv/Fm, including what we observed in the experiment, may not induce a prolonged impact to posttransplanting growth and development. The physiological damage at the seedling stage temporarily reduces the maximum photosynthetic capacity of the whole plant until new leaves are developed and damaged leaves are removed. For example, Smillie et al. (1988) reported cucumber, tomato, and 13 other species stored at 7 °C for 20 h had reduced Fv/Fm that persisted once plants were returned to 21 °C. Damaged tissues result in reduced capacity to combat oxidative stress, and an individual plant must devote more resources to prevent further physiological damage at the cost of its primary productivity. Regardless, advanced leaf injury, especially when visible on transplanting, would be perceived as low transplant quality for growers, especially when plants were produced from a third party.

Vegetative growth and floral development after transplanting.

Regardless of repeat or grafting, none of the vegetative growth parameters were affected by chilling treatments. Grafting affected the primary vine growth (cm/d) and was seen only in the first repeat (data not shown), resulting in increased length for grafted plants. Because of the greenhouse environmental conditions during spring vs. summer, all vegetable growth variables were more advanced in summer and exhibited greater extension growth (primary vine length and internode length) and leaf count in summer than spring, for both grafted and nongrafted plants (data not shown).

Days from transplant to the first male and female flower emergence was significantly affected by grafting (spring and summer) and chilling duration (spring only) where the number of days to reach first female flower anthesis linearly increased as chilling duration at 1 °C increased (Fig. 1C). Nongrafted plants had a greater extent of delay in first female flower development (i.e., a greater slope in the regression), indicating that they were more affected by chilling than grafted counterparts. Grafted and nongrafted plants exhibited delays in female flowering by up to 3.2 and 6.1 d, respectively, over nonchilled grafted and nonchilled nongrafted plants.

Delays in first flowering correlate well with delays in first harvest. For example, 1-week delay in flowering due to grafting is observed when watermelon is grafted onto bottle gourd rootstocks under nonoptimal growing conditions, and this grafting-delayed flowering can also delay fruit maturity by 1 week (Miguel, 1997). In our experiment, delay in flowering by grafting was observed also. This grafting-induced flowering delay is often attributed to the excessive vegetative growth, and recommendations are typically to manage vegetative vigor by optimizing nitrogen fertilization in the field. In contrast, delay in CIs received during seedling stage before transplanting may not be easily manageable, and therefore chilling stress must be avoided during transportation.

As briefly described earlier, differences between the two repeats in terms of chilling effects may be the difference in plant floral bud developmental stage at the time of chilling exposure. Watermelon seedlings begin to differentiate reproductive tissues in the meristem around the time of transplanting, based on preliminary meristem dissections we performed on seedlings. However, little is known about the impact of chilling on Cucurbitaceae (or Solanaceae) flower buds relative to their developmental stages. In contrast, more information is available for other fruit crops. For example, chilling of strawberry (Fragaria ×ananassa) plants (2 °C for 24 h) can decrease ovule viability, increase nonviable pollen, increase cell death, and disrupt development in four of 13 distinct stages of reproductive bud development (Ariza et al., 2015). In apricot trees, prebloom flower buds become susceptible to ice formation and bud drop (flower abortion), despite tolerating lower temperatures during overwintering (Julian et al., 2007). Based on preliminary flower dissections performed on nongrafted watermelon seedlings grown under similar conditions to spring, we were able to identify anthers, but not stigmas, present in the flower buds of some seedlings between 21 and 25 d after germination. The chilling treatments were conducted ≈28 d after germination in the present experiment, which suggests that differentiated flower parts were likely present at the time of chilling. Increased durations of 1 °C exposure during the seedling stage delayed flower emergence for both grafted and nongrafted plants in spring (Fig. 1C), but not in summer. This indicates that increasing chilling stress can cause disruption in reproductive development but may be due to the specific stage of flower development in the meristem. More research is needed to evaluate this theory, as well as confirming the rate of flower bud development under different environmental conditions and the sensitivity of reproductive developmental stages to chilling stress.

Expt. 2: Effects of varied CDHs at selected BT for plant response.

Like Expt. 1, no significant effects of chilling treatments were found in all posttransplanting growth and developmental parameters when conducted in summer months (data not shown). Therefore, the following data analysis and discussion were made using the repeat conducted during spring months.

Seedling visual quality relative to CDH.

After chilling, ΔMP ranged between a 4.1% and 23.4% increase among treatments (Table 1). Overall, ΔMP increased with either increasing duration of chilling exposure (as also seen in Expt. 1) or reducing the chilling temperature. Treatment III (0.4 °C for 44 h; Table 1) was the most damaging, yielding a ΔMP of 23.4%. When compared at a similar mean temperature (e.g., 0.4 to 0.5 °C, Treatment III vs. IV), longer duration of exposure tends to increase ΔMP. When compared at the same duration of exposure (e.g., Treatment V vs. VI) the temperature effect was not clear, likely due to the relatively large variability of temperature in this experiment.

When correlated with CDH computed with varied BT (3 to 15 °C), ΔMP showed a nonlinear response of increasing ΔMP with increasing CDH. Reduced mean square error (RMSE) found for each nonlinear regression analysis (between ΔMP and CDH) decreased as BT increases (data not shown). When 15 °C was selected as a BT (CDH15), ΔMP increased exponentially after 600 CDH15 (Fig. 2A). This indicates that visible seedling damage occurred at temperatures ≤15 °C but was not notable until after 600 CDH15 is reached. After this point, seedling damage rapidly increased. The most damaging chilling treatment (0.4 °C for 44 h, or 746 CDH15; Table 1) yielded a ΔMP of 23.4% of the true leaf area (Fig. 2A). Kozik and Wehner (2014) found that similar true leaf, growing point, and cotyledon damage to watermelon seedlings was accrued with 24 h of 2 °C storage as 36 h of 4 °C storage, indicating a similar facultative response (i.e., reduction in seedling quality) can be achieved with cumulative chilling at different temperatures and durations. Spalholz (2013) demonstrated two cultivars of watermelon seedlings can be stored at 5 °C with 2 µmol·m−2·s−1 PPFD and suffer no mortality after 2 weeks. However, one cultivar suffered 40% mortality after 3 weeks, and 100% of the seedlings from both cultivars had died after the fourth week. In this instance, seedling death resembled an exponential increase after 3 weeks. This study may indicate that a threshold of tolerance for chilling stress exists, but can vary depending on the conditions, cultivar, and measure of plant health (e.g., ΔMP and mortality). Possibly, this could be due to a reduced ability to remove harmful metabolites, enhanced production and reduced removal of reactive oxygen species, or a loss of critical structures that compromise tissues. These would result in increasing seedling injury and, eventually, death.

Fig. 2.
Fig. 2.

(A) ΔMP (percent of leaf area newly damaged) of seedlings evaluated 24 h after the chilling treatments with increasing chilling degree hours [base temperature (BT) = 15 °C] in Expt. 2 (spring) (n = 16 treatment containers). Means ± standard errors (n = 10 treatment containers). (B) Variable fluorescence/maximum fluorescence (Fv/Fm) ratios of seedlings across chilling degree hours (BT = 3) in Expt. 2 (spring). Means ± standard errors (n = 16). (C) Days from transplant to first female flower anthesis across chilling degree hours (BT = 4 °C) in Expt. 2 (spring). Means ± standard errors (n = 10 blocks).

Citation: HortScience 57, 8; 10.21273/HORTSCI16557-22

Chlorophyll fluorescence relative to CDH.

After the 72-h low-temperature treatments, the nonchilled control (72 h of 12 °C) had an Fv/Fm of 0.74 ± 0.01. The Fv/Fm decreased either as increasing duration of chilling exposure (as also seen in Expt. 1) or reducing the chilling temperature. The greatest reduction in Fv/Fm was found in the Treatment VI (1.2 °C for 48 h) to be 0.66 ± 0.04, which is an 11% reduction vs. the nonchilled control (Table 1).

When correlated with CDH, Fv/Fm showed a linear decrease with increasing CDH and had a smaller RMSE at lower BTs (data not shown). When 3 °C was selected as BT, the slope of linear equation showed that Fv/Fm decreased by 0.05 every increase of 100 CDH3 (Fig. 2B), which can be achieved for 50 h at 1 °C as an example. The same CDH3 induced similar Fv/Fm regardless of chilling temperature and exposure time. For example, Treatments II and IV (CDH3 = 87) or Treatments III and V (CDH3 = 124 or 125) had similar Fv/Fm (Table 1).

The regression analyses for Fv/Fm and CDH indicated the stronger correlations at lower BT in the temperature range we examined. This is an opposite trend observed for visual damage (as measured using ΔMP) where higher BT had a stronger correlation (i.e., BT = 15 °C). Further, although physical damage on true leaves (assessed by ΔMP) increased exponentially after a certain threshold was reached, the photosynthetic capacity of leaf tissue (assessed by Fv/Fm) linearly declined with CDH. Photosynthetic capacity declines as light-harvesting complexes are destabilized by low temperatures, and eventually reach a critical point in chilling accumulation that causes a more destructive tissue response (e.g., necrosis) (Lyons, 1973). Damaged tissues have reduced to no ability to harvest light energy, depending on the severity of damage. Reduction in Fv/Fm and visible seedling damage seen in this experiment was like that exhibited by seedlings in Expt. 1, exposed to 48 h of 1 °C chilling.

Vegetative growth and floral development after transplanting relative to CDH.

Vegetative growth was unaffected by any of the chilling treatments. Plants across all chilling treatments averaged 1.5 ± 0.04 cm/d of growth on the primary vine, 6.0 ± 0.07 cm internode lengths, and produced 0.3 ± 0.02 leaves/d, 0.03 ± 0.01 leaves/d, and 0.35 ± 0.03 leaves/d on the primary vine, secondary vine, and whole plant, respectively (data not shown).

Flower development was affected by the chilling treatments, but only for female flowers. Plants reached first male flower anthesis 42.1 ± 0.6 d after transplanting on average for all chilled treatments. Female flower emergence was delayed by either increasing duration of chilling exposure (as also seen in Expt. 1) or reducing the chilling temperature. The greatest delay was in Treatment III (0.4 °C for 44 h) and reached anthesis 4.3 d later than the control (Treatment VII; 11.8 °C for 72 h) (Table 1). Plants in the control (Treatment VII) reached anthesis before all other treatments, 34.6 ± 1.3 d after transplantation.

Significant linear correlations with CDH were found for days to first female flower. Regression analyses showed that RMSE was generally high and similar among the treatments due to relatively large variability (data not shown), but CDH and days to first female flower were found most highly correlated (lowest RMSE among the BT tested) with a BT of 4 °C. With a BT of 4 °C, increase in days (delay) of anthesis of first female flower was 2.6 d every 100 CDH4 (Fig. 2C), which can be achieved by 20 h at −1 °C for example. Similar CDH4 (Treatments III and V accumulating 168 and 169 CDH4, respectively) induced a similar delay (4.0–4.3 d), whereas the treatment temperature (0.4 vs. 0.8 °C) and duration (44 vs. 48 h) are different.

Previously, we exposed grafted and nongrafted watermelon seedlings to 0 to 48 h of 1 °C (Expt. 1 in this report) or 3 °C chilling (Ertle et al., 2021). There were no significant delays in flower anthesis in nongrafted watermelon plants when treated at 3 °C, whereas there were significant delays up to 6 d at 1 °C. Those two experiments were conducted using different cultivars (Tri-X-313 and SSX-8585) examined at 1 and 3 °C, respectively, and therefore the difference in results could be partially attributed to the possible cultivar difference. Nevertheless, when we attempt to apply the CDH4 to these earlier results, the longest duration (48 h) of 1 and 3 °C chilling accumulates 144 and 48 CDH4, respectively. We did not see any significant delay with less than 50 CDH4 in either Expts. 1 or 2, although accumulation greater than 50 CDH4 in either experiment correlated with delays in floweranthesis. Using CDH4, we further validated the model using existing data approximating the delay in female flower anthesis (Fig. 3). As plant development rate is highly affected by growing temperature conditions, the expected delays may need to be expressed with thermal time (e.g., degree days) instead of simple time. Nevertheless, there seems to be a threshold at ≈50 to 70 CDH4 to cause notable delay in female flower development and anthesis.

Fig. 3.
Fig. 3.

Delay (d) in female flower anthesis across chilling degree hours at 4 °C base temperature (CDH4) observed in four datasets when chilling was induced in the seedling stage. (A) Grafted and nongrafted watermelon plants reported by Ertle et al. (2021) with 0 to 48 h of exposure to 3 °C chilling. (B) Nongrafted watermelon plants from years 1 and 2 reported by Korkmaz and Dufault (2001) with 0 to 81 h of exposure to 2 °C chilling. (C) Grafted and nongrafted plants in the present Expt. 1 with 0 to 48 h of exposure to 1 °C chilling. (D) Nongrafted plants in the present Expt. 2 with 24 to 48 h of exposure to −0.4 to 1.2 °C chilling.

Citation: HortScience 57, 8; 10.21273/HORTSCI16557-22

Korkmaz and Dufault (2001) exposed diploid watermelon seedlings to 2 °C for between 9 and 81 h, and found that seedling quality and posttransplanting growth in an open field was negatively affected with increased chilling. After transplanting, seedlings exposed to longer durations of 2 °C chilling took a longer time to produce secondary vines, reach flower anthesis, and fruit set. We plotted their reported 2-year data of delayed female flower anthesis against our CDH4 model (Fig. 3B). We found that in both years, anthesis was delayed with increasing CDH4, and the trend was mostly linear as CDH4 accumulated. Linear trendlines over each dataset yielded slopes between 0.023 and 0.050. Thus, for every 50 CDH4 accumulated, female flower anthesis is delayed between 1.1 and 3.7 d for nongrafted plants, and 1.9 d for grafted plants. The slopes for both years of Korkmaz and Dufault (2001) data fell between the slopes derived from our data for nongrafted plants in Expt. 1 and Expt. 2, indicating our model can predict flowering delays with a reasonable accuracy. Although Korkmaz and Dufault (2001) used diploid seedlings, we found our results using triploid seedlings to exhibit similar flowering delays. No data are available comparing chilling tolerance of diploid vs. triploid watermelon seedlings, but the similarity in trends between our results and their study indicate that there is little difference.

Conclusions

Grafted and nongrafted watermelon seedling transplants could experience significant delays in floral development in excess of 6 d with up to 48 h of 1 °C exposure, with decreased seedling visual quality and chlorophyll fluorescence. However, grafting mitigated the degree of flowering delay occurring after exposure to the same amount of chilling at 1 °C. Summer growing conditions also masked the posttransplanting delay in reproductive development seen in spring. Our experiment examining combinations of chilling length (between 24 and 48 h) and temperature (between −0.4 and 1.2 °C) showed that acute chilling stress response of watermelon seedlings could be modeled using CDH computed with specific BT. We found that BTs that most accurately predict specific plant responses to chilling varied (from as high as 15 °C for visual quality to as low as 3 or 4 °C for Fv/Fm and first female flower development, respectively). Although seedling damage and Fv/Fm were reduced with accumulating CDH, the most significant impact of delay in female flower development occurred after 50 to 70 CDH4. Therefore, if temperatures during transportation fall below 4 °C, measures should be taken so that CDH4 does not accumulate greater than 50 to 70, which will delay female flowering predictably beyond this threshold according to our model. Further study is needed to better understand the specific influences of chilling stress during the seedling stage and examine the physiological impacts on reproductive development.

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Contributor Notes

C.K. is the corresponding author. E-mail: Kubota.10@osu.edu.

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    Fig. 1.

    (A) Percent change in leaf tissue damage (ΔMP) of grafted and nongrafted seedlings in Expt. 1 (spring), 24 h after the end of chilling as affected by the duration of exposure to 1 °C. Means ± standard errors (n = 5 treatment containers). (B) Chlorophyll fluorescence parameter variable fluorescence/maximum fluorescence (Fv/Fm) as affected by duration of exposure to 1 °C in Expt. 1 (summer). Data of grafted and nongrafted plants were combined, as there was no significant effect of grafting on Fv/Fm. Means ± standard errors (n = 4 treatment containers). (C) Days from transplant to first female flower anthesis for grafted and nongrafted plants as affected by durations of exposure to 1 °C in Expt. 1 (spring). Means ± standard errors (n = 10 blocks).

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    Fig. 2.

    (A) ΔMP (percent of leaf area newly damaged) of seedlings evaluated 24 h after the chilling treatments with increasing chilling degree hours [base temperature (BT) = 15 °C] in Expt. 2 (spring) (n = 16 treatment containers). Means ± standard errors (n = 10 treatment containers). (B) Variable fluorescence/maximum fluorescence (Fv/Fm) ratios of seedlings across chilling degree hours (BT = 3) in Expt. 2 (spring). Means ± standard errors (n = 16). (C) Days from transplant to first female flower anthesis across chilling degree hours (BT = 4 °C) in Expt. 2 (spring). Means ± standard errors (n = 10 blocks).

  • View in gallery
    Fig. 3.

    Delay (d) in female flower anthesis across chilling degree hours at 4 °C base temperature (CDH4) observed in four datasets when chilling was induced in the seedling stage. (A) Grafted and nongrafted watermelon plants reported by Ertle et al. (2021) with 0 to 48 h of exposure to 3 °C chilling. (B) Nongrafted watermelon plants from years 1 and 2 reported by Korkmaz and Dufault (2001) with 0 to 81 h of exposure to 2 °C chilling. (C) Grafted and nongrafted plants in the present Expt. 1 with 0 to 48 h of exposure to 1 °C chilling. (D) Nongrafted plants in the present Expt. 2 with 24 to 48 h of exposure to −0.4 to 1.2 °C chilling.

  • Allen, D.J. & Ort, D.R. 2001 Impacts of chilling temperatures on photosynthesis in warm-climate plants Trends Plant Sci. 6 1 36 42 https://doi.org/10.1016/S1360-1385(00)01808-2

    • Search Google Scholar
    • Export Citation
  • Ariza, M.T., Soria, C. & Martınez-Ferri, E. 2015 Developmental stages of cultivated strawberry flowers in relation to chilling sensitivity AoB Plants 7 plv012 https://doi.org/10.1093/aobpla/plv012

    • Search Google Scholar
    • Export Citation
  • Bock, C.H., Gottwald, T.R., Parker, P.E., Ferrandino, F., Welham, S., van den Bosch, F. & Parnell, S. 2010 Some consequences of using the Horsfall-Barratt scale for hypothesis testing Phytopathology 100 1030 1041

    • Search Google Scholar
    • Export Citation
  • Cantliffe, D.J. 1993 Pre- and postharvest practices for improved vegetable transplant quality HortTechnology 3 4 415 418 https://doi.org/10.21273/HORTTECH.3.4.415

    • Search Google Scholar
    • Export Citation
  • Darré, M., Valerga, L., Zaro, M.J., Lemoine, M.L., Concellón, A. & Vicente, A.R. 2022 Eggplant grafting on a cold-tolerant rootstock reduces fruit chilling susceptibility and improves antioxidant stability during storage J. Sci. Food Agr. 102 8 3350 3358 https://doi.org/10.1002/jsfa.11682

    • Search Google Scholar
    • Export Citation
  • Davis, A.R., Perkins-veazie, P., Sakata, Y., Maroto, J.V., Lee, S., Huh, Y., Sun, Z., Miguel, A., King, S.R., Cohen, R. & Lee, J. 2008 Cucurbit grafting Crit. Rev. Plant Sci. 27 50 74 https://doi.org/10.1080/07352680802053940

    • Search Google Scholar
    • Export Citation
  • Ding, M., Bie, B., Jiang, W., Duan, Q., Du, H. & Huang, D. 2011 Physiological advantages of grafted watermelon (Citrullus lanatus) seedlings under low-temperature storage in darkness HortScience 46 7 993 996 https://doi.org/10.21273/hortsci.46.7.993

    • Search Google Scholar
    • Export Citation
  • Elwakil, W.M., Dufault, N.S., Freeman, J.H. & Mossler, M. 2017 Florida crop/pest management profile: Watermelon Univ. of Fl. Inst. of Food and Agr. Sci Electronic Data Info. Source CIR1236:1–27. 2 Feb. 2020. <https://edis.ifas.ufl.edu/pi031>

    • Search Google Scholar
    • Export Citation
  • Erez, A., Fishman, S., Linsley-Noakes, G.C. & Allan, P. 1990 The dynamic model for rest completion in peach buds Acta Hort. 276 165 174 https://doi.org/10.17660/ActaHortic.1990.276.18

    • Search Google Scholar
    • Export Citation
  • Ertle, J. & Kubota, C. 2020 North American grafting survey Ohio State Univ. Dep. Hort. and Crop Sci., Columbus, OH. 1 Apr. 2022. <https://u.osu.edu/cepptlab/extension/>

    • Search Google Scholar
    • Export Citation
  • Ertle, J., Kubota, C. & Pliakoni, E. 2021 Transplant quality and growth of grafted and non-grafted watermelon seedlings as affected by chilling during simulated long-distance transportation Acta Hort. 1302 87 94 https://doi.org/10.17660/actahortic.2021.1302.12

    • Search Google Scholar
    • Export Citation
  • Horsfall, J.G. & Barratt, R.W. 1945 An improved grading system for measuring plant disease Phytopathology 35 655

  • Hou, W., Sun, A.H., Chen, H.L., Yang, F.S., Pan, J.L. & Guan, M.Y. 2016 Effects of chilling and high temperatures on photosynthesis and chlorophyll fluorescence in leaves of watermelon seedlings Biol. Plant. 60 1 148 154 https://doi.org/10.1007/s10535-015-0575-1

    • Search Google Scholar
    • Export Citation
  • Janmohammadi, M. 2012 Metabolomic analysis of low temperature responses in plants Curr. Opin. Agr. 1 1 1 6

  • Julian, C., Herrero, M. & Rodrigo, J. 2007 Flower bud drop and pre-blossom frost damage in apricot (Prunus armeniaca L.) J. Appl. Bot. Food Qual. 81 21 25

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